Method and compositions for improved lignocellulosic material hydrolysis

ABSTRACT

A method of digesting a lignocellulosic material is disclosed. In one embodiment, the method comprises the step of exposing the material to an effective amount of Streptomyces sp. ActE secretome such that at least partial lignocellulosic digestion occurs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit from U.S. Provisional Application 61/579,301 filed Dec. 22, 2011 and U.S. Provisional Application 61/579,897 filed Dec. 23, 2011, both of which are incorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DE-FC02-07ER64494 awarded by the US Department of Energy and GM094584 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Cellulose is the most abundant organic polymer on Earth and represents a vast source of renewable energy. Most of this energy is stored in the recalcitrant polysaccharide cellulose, which is difficult to hydrolyze because of the highly crystalline structure, and in hemicellulose, which presents challenges because of its structural diversity and complexity. Plant cell walls are approximately composed in pinewood of lignin (30% by weight), hemicellulose (glucomannan, 20%, arabinoxylan, 10%), and crystalline cellulose (40%), which presents a major barrier to efficient use. In terrestrial ecosystems, cellulolytic microbes help drive carbon cycling through the deconstruction of biomass into simple sugars. The deconstruction is largely accomplished through the action of combinations of secreted glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), and carbohydrate binding modules (CBMs) (Baldrian and Valaskova, 2008; Cantarel, et al., 2009; Lynd, Weimer, et al., 2002; Schuster and Schmoll, 2010). Consequently, organisms from many lignocellulose-rich environments and their enzymes are being studied for new insights into overcoming this barrier.

In order to obtain the hydrolysis of crystalline cellulose, enzymes must cleave three types of glycosidic bonds. These enzymes are endocellulases, which cleave beta-1,4 glycosidic bonds that reside within intact cellulose strands in the crystalline face, non-reducing-end exocellulases, which remove cellobiose units from the non-reducing end of cellulose strands, and reducing-end exocellulases, which remove glycosyl units from the reducing-end of a cellulose strand. The endocellulolytic reaction is essential because it creates the non-reducing and reducing ends that serve as the starting point for exocellulolytic reactions. The exocellulolytic reactions are essential because they remove glycosyl groups in a processive manner from the breakages in the cellulose strand introduced by the endocellulases, thus amplifying the single initiating reaction of the endocellulases.

Trichoderma reesei and Clostridium thermocellum are well-characterized cellulose-utilizing organisms (Merino and Cherry, 2007; Bayer et al., 2008; Wilson, 2011). T. reesei is a slow-growing eukaryote fungus that secretes enzymes containing glycoside hydrolase (GH) domains fused to carbohydrate binding domains, while C. thermocellum is a strictly anaerobic prokaryote that predominantly assembles GHs and carbohydrate-binding molecules (CBMs) into a large complex called the cellulosome. Enzymes from these free-living organisms cleave polysaccharides using general acid-base catalyzed hydrolytic reactions (Vuong and Wilson, 2010). Moreover, fungal and microbial communities associated with termites (Scharf et al., 2011) shipworms (Luyten et al., 2006), and rumen (Hess et al., 2011) contribute these types of hydrolytic enzymes to their respective anaerobic niches.

Some free-living aerobes such as Cellvibrio japonicus (Ueda 107) (DeBoy et al., 2008), Streptomyces (Schlochtermeier et al., 1992; Wilson, 1992; Forsberg et al., 2011), Thermoascus aurantiacus (Langston et al., 2011; Quinlan et al., 2011) and Serratia marcescens (Vaaje-Kolstad et al., 2010) also grow on biomass polysaccharides. Recent work with some of these organisms has identified that the structurally related fungal GH61 (Langston et al., 2011; Quinlan et al., 2011) and bacterial CBM33 (Forsberg et al., 2011) families of proteins catalyze a previously unrecognized oxidative breakage of glycosidic bonds. This reaction is thought to be an endo-cleavage, with the oxidation reaction yielding gluconate and keto-sugars instead of the typically observed reducing and non-reducing sugars obtained from hydrolytic cellulases.

Actinobacteria in the genus Streptomyces are an ecologically important group, especially in soil environments, where they are considered to be vital players in the decomposition of cellulose and other biomass polymers (Cantarel et al., 2009; Crawford et al., 1978; Goodfellow and Williams, 1983; McCarthy and Williams, 1992). Streptomyces are able to utilize a wide range of carbon sources, form spores when resources are depleted, and produce antimicrobial secondary metabolites to reduce competition (Goodfellow and Williams, 1983; Schlatter et al., 2009).

Although a large number of Streptomyces species can grow on biomass, only a small percentage (14%) have been shown to efficiently degrade crystalline cellulose (Wachinger, Bronnenmeier, et al., 1989). Furthermore, the secreted cellulolytic activities of only a few species have been biochemically characterized, and still fewer species have been examined to identify key biomass degrading enzymes (Ishaque and Kluepfel, 1980; Semedo et al., 2004). Streptomyces reticuli is one of the best-studied cellulose- and chitin-degrading soil-dwelling Streptomyces; functional analyses of several important cellulases and other hydrolytic enzymes have been reported (Wachinger, Bronnenmeier, et al., 1989; Schlochtermeier, Walter, et al., 1992; Walter and Schrempf, 1996).

Furthermore, polysaccharide monooxygenase (PMO) activity with cellulose was identified using the CBM33 protein from Streptomyces coelicolor (Forsberg, et al., 2011), which suggests Streptomyces may use both hydrolytic and oxidative enzymes to deconstruct biomass. With the tremendous amount of sequence data collected in the past few years, and despite the view that Streptomyces make important contributions to cellulose degradation in the soil, genome-wide analyses of cellulolytic Streptomyces have not been reported.

In addition to their putative roles in carbon cycling in the soil, Streptomyces may also potentiate biomass deconstruction in insects through symbiotic associations (Bignell, Anderson, et al., 1991; Pasti and Belli, 1985; Pasti, Pometto, et al., 1990; Schafer, et al., 1996). Recent work has identified cellulose degrading Streptomyces associated with the pine-boring woodwasp Sirex noctilio, including Streptomyces sp. SirexAA-E (ActE) (Adams, et al., 2011). S. noctilio is a highly destructive wood-feeding insect that is found throughout forests in Eurasia and North Africa and is spreading invasively in North America and elsewhere (Bergeron, et al., 2011). While the wasp itself does not produce cellulolytic enzymes, evidence supports the role of a symbiotic microbial community that secretes biomass-degrading enzymes to facilitate nutrient acquisition for developing larvae in the pine tree (Kukor and Martin, 1983).

The white rot fungus, Amylostereum areolatum, is the best-described member of this community, and the success of Sirex infestations is thought to arise from the insect's association with this cellulolytic fungal mutualist. However, work with pure cultures has suggested that ActE and other Sirex-associated Streptomyces are more cellulolytic than A. areolatum (Adams, et al., 2011).

Optimal activity in the CBM33 enzymes apparently requires the addition of a transition metal ion such as Cu(II), Fe(III), or Mn(II) and an external reducing agent. In the laboratory, the reducing agent can be provided by ascorbate. In natural systems, the reducing function is most likely provided by another redox active protein such as cellobiose dehydrogenase (Langston et al., 2011; Quinlan et al., 2011) or some other presently unknown protein.

Needed in the art are improved compositions and organisms for digestion of lignocellulosic materials.

BRIEF SUMMARY

The invention relates generally to methods and compositions for digesting lignocellulosic material and more particularly to methods that involve exposing the material to secretome derived from Streptomyces sp. ActE.

In a first aspect, the present invention is summarized as a method of digesting a lignocellulosic material comprising the step of exposing the material to an effective amount of Streptomyces sp. ActE secretome preparation such that at least partial lignocellulosic digestion occurs.

In some embodiments of the first aspect, the preparation is a supernatant preparation obtained from a Streptomyces sp. ActE culture. In some embodiments of the first aspect, the preparation is obtained from Streptomyces sp. ActE grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass and chitin. In some embodiments of the first aspect, the lignocellulosic material is selected from the group consisting of materials that comprise at least 75% cellulose, cellulose/hemicelluloses, xylose, biomass and chitin.

In a second aspect, the present invention is summarized as a purified preparation comprising the Streptomyces sp. ActE secretome.

In some embodiments of the second aspect, the preparation is a supernatant preparation obtained from a Streptomyces sp. ActE culture. In some embodiments of the second aspect, Streptomyces sp. ActE is grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass and chitin.

In a third aspect, the present invention is summarized as a composition useful for digesting lignocellulosic material comprising SActE_0237 (GH6) (SEQ ID NO:1) gene or expression product thereof.

In a fourth aspect, the present invention is summarized as a composition useful for digesting lignocellulosic material comprising SActE_0236 (GH48) (SEQ ID NO:2) gene or expression product thereof.

In a fifth aspect, the present invention is summarized as a composition useful for digesting lignocellulosic material comprising SActE_3159 (CBM33) (SEQ ID NO:3) gene or expression product thereof.

In a sixth aspect, the present invention is summarized as a composition useful for digesting lignocellulosic material comprising SActE_0482 (GH5) (SEQ ID NO:4) gene or expression product thereof.

In a seventh aspect, the present invention is summarized as a composition useful for digesting lignocellulosic material comprising SActE_0265 (GH10) (SEQ ID NO:5) gene or expression product thereof.

In a eighth aspect, the present invention is summarized as a composition useful for digesting lignocellulosic material comprising SActE_2347 (GH5) (SEQ ID NO:6) gene or expression product thereof.

In a ninth aspect, the present invention is summarized as a composition useful for digesting lignocellulosic material comprising SActE_0237 (GH6) (SEQ ID NO:1), SActE_0236 (GH48) (SEQ ID NO:2), SActE_3159 (CBM33)(SEQ ID NO:3), SActE_0482 (GH5) (SEQ ID NO:4) and gene or expression product thereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for cellulose utilization. In these embodiments the composition can additionally comprise at least one member selected from SActE_0265 (GH10) (SEQ ID NO:5) and SActE_2347 (GH5) (SEQ ID NO:6) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for xylan release. By “release,” we mean degradation, such as hydrolysis, and release of an important or desired product. In these embodiments the composition can additionally comprise at least one member selected from SActE_0265 (GH10) (SEQ ID NO:5), SActE_0358 (GH11) (SEQ ID NO:8), SActE_0357 (CE4) (SEQ ID NO:7), SActE_5978 (PL1)(SEQ ID NO:16) and SActE_5230 (xylose isomerase) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for chitin release. In these embodiments the composition can additionally comprise at least one member selected from SActE_4571 (GH18), SActE_2313 (CBM33), SActE_4246 (GH18), SActE_3064 (GH19) and SActE_5764 (GH18) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for biomass degradation. In these embodiments the composition can additionally comprise SActE_5457 (GH46) (SEQ ID NO:14) gene or expression products thereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for mannan release. In these embodiments the composition can additionally comprise SactE_2347 (GH5) (SEQ ID NO:6) gene or expression products thereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for beta-1,3-glucan release. In these embodiments the composition can additionally comprise at least one member selected from SActE_4755 (GH64) (SEQ ID NO:13) and SActE_4738 (GH16) (SEQ ID NO:12) genes or expression products thereof. In a preferred embodiment, the composition comprises both of the genes or expression products.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for pectin cleavage. In these embodiments the composition can additionally comprise SActE_1310 (PL3) (SEQ ID NO:9) gene or expression products derived thereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for alginate release. In these embodiments the composition can additionally comprise SActE_4638 (SEQ ID NO:11) gene or expression products derived thereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth, and ninth aspects, the composition is optimized for galactose release. In these embodiments the composition can additionally comprise SactE_5647 (GH87) (SEQ ID NO:15) gene or expression products derived thereof.

In a tenth aspect, the present invention is summarized as a composition useful for xylan degradation comprising SActE_0265 (GH10) (SEQ ID NO:5) and SActE_0358 (GH11) (SEQ ID NO:8) gene or expression products thereof.

In some embodiments of the tenth aspect, the composition additionally comprises SActE_0265 (GH10) (SEQ ID NO:5), SActE_0358 (GH11) (SEQ ID NO:8), SActE_0357 (CE4) (SEQ ID NO:7), SActE_5978 (PL1) (SEQ ID NO:16), and SActE_5230 (xylose isomerase) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In an eleventh aspect, the present invention is summarized as a composition useful for biomass degradation comprising SActE_0237 (GH6) (SEQ ID NO:1), SActE_0482 (GH5) (SEQ ID NO:4), SActE_3159 (CBM33)(SEQ ID NO:3), SActE_0236 (GH48) (SEQ ID NO:2), SActE_3717 (GH9) (SEQ ID NO:10), SActE_0265 (GH10) (SEQ ID NO:5), SActE_0358 (GH11) (SEQ ID NO:8), SActE_2347 (GH5) (SEQ ID NOs:6) and SActE_1310 (PL1) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In a twelfth aspect, the present invention is summarized as a composition useful for cellulose degradation comprising SActE_0237 (GH6) (SEQ ID NO:1), SActE_0482 (GH5) (SEQ ID NO:4), SActE_3159 (CBM33) (SEQ ID NO:3) SActE_0236 (GH48) (SEQ ID NO:2), SActE_2347 (GH5) (SEQ ID NO:6), and SActE_0265 (GH10) (SEQ ID NO:5) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In a thirteenth aspect, the present invention is summarized as a method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of any one of the third to eighth aspects of the invention, wherein the exposed material is at least partially digested.

In a fourteenth aspect, the present invention is summarized as a purified preparation of Streptomyces sp. ActE, wherein the Streptomyces sp. ActE has been grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass, and chitin.

In a fifteenth aspect, the present invention is summarized as a purified preparation of Streptomyces sp. ActE, wherein the Streptomyces sp. ActE has been grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon in the substrate is derived from pretreated lignocellulosic material.

In some embodiments of the fifteenth aspect, the pretreated material has been exposed to pretreatment selected from the group consisting of acid hydrolysis, steam explosion, ammonia fiber expansion (AFEX), organosolve, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), ionic liquids, metal-catalyzed hydrogen peroxide, alkaline wet oxidation and ozone pretreatment. In some embodiments of the fifteenth aspect, the pretreated material is wood.

These and other features, objects, and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The present invention will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

FIG. 1 is a set of pictures showing growth of ActE in minimal medium containing filter paper as the sole carbon source. (A) Growth of ActE, Streptomyces coelicolor, and Streptomyces griseus in minimal medium for 7 days at 30° C. and pH 6.9. The expanded image shows small colonies of S. coelicolor and S. griseus forming on the surface of the paper. (B) Growth of ActE and Trichoderma reesei Rut-C30 for 7 days at 30° C. and pH 6.0.

FIG. 2 is a set of graphs demonstrating reactions of ActE secretomes and Spezyme CP. (A) HPLC of sugars released from cellulose (1, cellotriose; 2, cellobiose; 3, glucose) and quantification of glucose equivalent (insert). (B) Reducing sugars released from xylan and mannan by the secretomes of ActE grown on cellulose and xylan. (C) Total reducing sugar released from ionic liquid-switchgrass (IL-SG) or AFEX-switchgrass (AFEX-SG) in reactions of the ActE cellulose, AFEX-SG, and IL-SG secretomes and Spezyme CP. Data represent the mean±s.d. from three experiments; * indicates P<0.01 compared with Spezyme CP.

FIG. 3 is a table illustrating composition of ActE secretomes identified by LC-MS/MS. (A) CAZy genes account for 2.6% of the 6357 predicted protein-coding sequences in the ActE genome. (B) Identity of most abundant proteins in the cellulose secretome proteins is sorted according to decreasing spectral counts (accounting for 95% of total spectral counts); corresponding spectral counts from other secretomes are also shown.

FIG. 4 is a systematic diagram showing genome-wide changes in expression during growth of ActE on AFEX-treated switchgrass (AFEX-SG) versus glucose. Nodes are genes (circles) or KEGG/CAZy functional categories (yellow triangles); edges indicate that the gene belongs to the indicated functional group as defined by either KEGG or CAZy analysis. Gene node sizes reflect expression intensity determined by microarray from growth on AFEX-SG as a log₂ ratio, where the genome-wide average transcriptional intensity was ˜10.5 for both substrates. Node colors represent expression changes as the log₂ ratio of AFEX-SG/glucose transcript intensities.

FIG. 5 is a diagram with a table showing expression of ActE CAZy genes on various carbon sources. (A) Hierarchical clustering of expression for 167 CAZy genes from the ActE genome during growth on the indicated substrates. (B) Identity of CAZy genes with distinct changes in expression observed in group 1 CAZy genes during growth in different carbon sources.

FIG. 6 is a set of scanning electron microscopy (SEM) images showing ActE grown on different carbon sources including glucose, cellulose, xylan, switchgrass, ammonia fiber expansion-treated switchgrass (AFEX-SG) and ionic liquid-treated switchgrass (IL-SG). ActE cells were grown in minimum medium with the indicated substrate as a sole carbon source for 7 days at 30° C. The scale bar indicates 5 μm.

FIG. 7 is a set of graphs demonstrating fractionation of the ActE cellulose secretome and assays of reactions with different polysaccharides. (A) Anion exchange chromatography was performed using the ActE cellulose secretome, and fractions were collected and analyzed by SDS-PAGE. Lowercase letters indicate protein identified by MALDI-TOF MS shown in FIG. 17. (B) Results from hydrolysis assays for reaction with filter paper (FP), xylan, mannan and beta-1,3 glucan as detected by DNS assay of each fraction. The percentage reactivity relative to the maximum activity observed for each substrate is shown. Error bars indicate the standard deviation, with n=3 for technical replicates.

FIG. 8 a set of diagrams showing temperature and pH profiles of the ActE secretome obtained from growth on AFEX-treated corn stover. (A) The effect of temperature on the deconstruction of AFEX-treated switchgrass (AFEX-SG) and ionic liquid-treated switchgrass (IL-SG). The relative activity of the ActE secretome was compared to the maximal rates determined for reaction with AFEX-SG (blue star), and IL-SG (red star) at pH 6.0. (B) The effect of pH on the AFEX-SG and IL-SG deconstruction activities in the indicated ActE secretomes. The maximal rates observed for AFEX-SG and IL-SG were at pH 7.0 (blue star) and pH 8 (red star), respectively. Reactions were carried out at 40° C. and the 0.1 M buffers used were citrate (pH 4.5), phosphate (pH 6-8), CHES (pH 9-10), and CAPS (pH 11). The reaction was performed for 20 h and the reducing sugar content was measured by DNS assay.

FIG. 9 is a systematic diagram showing genome-wide changes in expression during growth of ActE on substrate cellobiose versus glucose visualized as a Cytoscape interaction network. Nodes are genes (circles) or KEGG/CAZy functional categories (yellow triangles); edges indicate that the gene belongs to the indicated functional group as defined by either KEGG or CAZy analysis. Gene node sizes reflect expression intensity determined by microarray from growth on substrate as a log 2 ratio. Node colors represent expression changes as the log 2 ratio of substrate/glucose transcript intensities, where the genome-wide average transcriptional intensity was ˜10.5 for both substrate and glucose. Transcripts with less than two-fold changes in expression intensity are colored white; transcripts with greater than two-fold increase in expression intensity during growth on substrate are shown as a red gradient; transcripts with greater than two-fold increase in expression intensity during growth on glucose are shown as a blue gradient.

FIG. 10 is a systematic diagram showing genome-wide expression changes for growth on the substrate cellulose versus glucose visualized as a Cytoscape interaction network. Other information is the same as that described in FIG. 9.

FIG. 11 is a systematic diagram showing genome-wide expression changes for growth on the substrate xylan versus glucose visualized as a Cytoscape interaction network. Other information is the same as that described in FIG. 9.

FIG. 12 is a systematic diagram showing genome-wide expression changes for growth on the substrate switchgrass versus glucose visualized as a Cytoscape interaction network. Other information is the same as that described in FIG. 9.

FIG. 13 is a systematic diagram showing genome-wide expression changes for growth on the substrate IL-treated switchgrass versus glucose visualized as a Cytoscape interaction network. Other information is the same as that described in FIG. 9.

FIG. 14 is a systematic diagram showing genome-wide expression changes for growth on the substrate chitin versus glucose visualized as a Cytoscape interaction network. Other information is the same as that described in FIG. 9.

FIG. 15 is a diagram with a table showing expression of 167 predicted CAZy genes in ActE, highlighting group 2 genes. These genes showed no signal above the average genomic expression intensity (log 2=10.5). (A) Clustering of genes with similar expression profiles. (B) Additional information on group 2 genes including expression profile, SACTE_locus ID, CAZy family, and annotated function.

FIG. 16 is a diagram with a table showing expression of 167 predicted CAZy genes in ActE, highlighting group 3 genes. (A) Clustering of genes with similar expression profiles. (B) Additional information on group 3 genes including expression profile, SACTE_locus ID, CAZy family, and annotated function.

FIG. 17 is a table illustrating proteins separated by ion exchange chromatography and identified by mass spectrometry.

FIG. 18 is a table showing spectra count of proteins identified on each substrate, where top 95% spectra covered were highlighted green, light purple, purple, blue, orange, pink, light blue and yellow on glucose, cellobiose, cellulose, xylan, switchgrass, AFEX-SG, IL-SG and chitin, respectively.

FIG. 19 shows the nucleic acid sequences of the ActE genes.

FIG. 20 shows the amino acid sequences of the ActE genes.

FIG. 21 is a graph illustrating a comparison of specific activities of Streptomyces sp. ActE secretomes with Spezyme CP. FIG. 21A depicts relative specific activity of ActE secretomes prepared from growth on cellulose or xylan and Spezyme CP (100%) for reducing sugar release from xylan or mannan. FIG. 21B depicts relative activity (pH 6.0, 40° C.) of ActE cellulose secretome and CelLcc_CBM3a, an engineered C. thermocellum endo/exoglucanase, compared to Spezyme CP. Total amounts of protein included in all reactions were equivalent.

FIG. 22 illustrates nucleotide (SEQ ID NO:63) and amino acid (SEQ ID NO:64)sequence of CelLcc_CBM3a. Construct described in US Patent Application Publication No.: US2010/037094 (Fox and Elsen).

FIG. 23 is a graph illustrating SDS-PAGE of Streptomyces sp. ActE secretomes obtained from growth on minimal medium containing different substrates (SG, switchgrass; CS, corn stover; UBLPKP, unbleached lodgepole pine kraft pulp; BSKP, bleached spruce kraft pulp; LP-SPORL, lodgepole pine pretreated by sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL)). Culture secretomes were separated after 7 days of growth at 30° C. by centrifugation and concentrated by ultrafiltration. Sample loading was normalized to total protein. The identities of proteins were determined from samples extracted from the SDS-PAGE gel. Among the 162 proteins accounting for 95% of spectral counts from the glucose secretome, most were intracellular proteins originating from cell lysis during growth, and were not detected in the polysaccharide secretomes.

FIG. 24 is a graph illustrating SDS-PAGE of time-dependent changes in the Streptomyces sp. ActE secretome obtained from growth on minimal medium containing cellulose. Culture secretomes were collected after 7 days by centrifugation and concentrated by ultrafiltration. The concentrated secretomes were incubated at 25° C. for the indicated times and analyzed. Protein bands with time-dependent decrease in intensity were excised from the gel and identified by LC-MS/MS.

FIG. 25 illustrates synergy of recombined fractions from ion exchange chromatography. All reactions were prepared to contain the same total amount of protein.

FIG. 26 is a set of graphs illustrating mannanase activity demonstrated in fractions containing various naturally truncated versions of SACTE_2347 (GH5). FIGS. 26A-B depict proteins found in previous assayed fractions. FIG. 26C depicts Coomassie Blue staining of 12% polyacrylamide gel (PAGE) separation of different mannanase isoforms. Three polypeptide bands corresponding to SACTE_2347 (GH5) with molecular masses of ˜57, ˜45, and ˜37 kDa. FIG. 26D depicts a zymogram performed in the presence of 0.5% mannan. The strong clearing zone in fraction F1 associated with the ˜37 kDa isoform demonstrates how size reduction can increase the specific activity of a protein.

FIG. 27 is a set of graphs illustrating ion exchange fractionation of Streptomyces sp. ActE secretome. FIG. 27A depicts an SDS-PAGE analysis of the fractionation of an ActE secretome by ion exchange chromatography. FIG. 27B depicts catalytic assays of the separate fractions at 40° C. for 20 h in 0.1 M phosphate buffer, pH 6.0, showing different enzymes are capable of reacting with xylan, mannan, and cellulose. The reactivity of fractions marked with stars is also described in FIG. 25A.

FIG. 28 is a SDS-PAGE graph and a list illustrating mass spectral assignment of polypeptides from the Streptomyces sp. ActE secretome separated by ion exchange chromatography. FIG. 28A depicts an SDS PAGE of separated fractions annotated with identities of polypeptides determined by LC-MS analysis. FIG. 28B depicts information on the identified proteins including gene locus, function, CAZy GH and CBM assignments, number of amino acid (AA) residues, and best BLAST result for relationship to another known enzyme. The reactivity of fractions marked with stars is also described in FIG. 25A.

FIG. 29 is a SDS-PAGE graph and a table that demonstrates the existence of xylanases from Streptomyces sp. ActE. Five ActE proteins were produced using cell-free translation as described in US Patent Application Publication No.: US2010/037094 (Fox and Elsen). FIG. 29A depicts a stain-free gel image of proteins produced by wheat germ cell-free translation (indicated by asterisks). FIG. 29B depicts a summary of protein information, expression and secretion data, and diagnostic assay results. Small molecule assays (MUG, methylumbelliferyl glucoside; MUC, methylumbelliferyl cellobioside; MUM, methylumbelliferyl mannoside and MUX2, methylumbelliferyl xylobioside) were performed in 0.1 M phosphate buffer, pH 6.0, at 30° C. SACTE_0265 and SACTE_0358, highly expressed and secreted proteins during growth on xylan, are confirmed by these assays to be xylanases. Results from three other non-secreted ActE enzymes are provided as controls.

FIG. 30 is a graph illustrating quantification of total secreted protein obtained from Streptomyces sp. ActE grown on different substrates (AFEX-CS, AFEX corn stover; UBLPKP, unbleached lodgepole pine kraft pulp; BSKP, bleached spruce kraft pulp; LP-SPORL, lodgepole pine pretreated by SPORL).

FIG. 31 is a graph illustrating the temperature versus activity profile of the Streptomyces sp. ActE secretome obtained from growth on cellulose. Hydrolysis activities were measured by DNS assay. Greater than 80% of maximal rates for cellulase and mannase activity were observed at the range of 31-43° C., while greater than 80% of maximal rate for xylanase activity was observed in the range of 35-59° C.

FIG. 32 is a graph illustrating the pH versus activity profile of the Streptomyces sp. ActE secretome obtained from growth on cellulose. The maximal rate was observed at approximately pH 6. Buffers used in this study were 0.1 M citrate (pH 4.5), phosphate (pH 6-8), CHES (pH 9-10) and CAPS (pH 11).

FIG. 33 is a SDS-PAGE graph illustrating ActE induction in medium containing as little as 20% cellulose.

FIG. 34 is a set of Venn diagrams representing 95% of total proteins identified in LC-MS/MS analyses generated using VennMaster-0.37.5 (Kestler et al., 2008). FIG. 8A depicts secretomes obtained from growth on glucose, Sigmacell™, and xylan. FIG. 8B depicts secretomes obtained from growth on switchgrass, ammonia fiber expansion (AFEX)-SG, and IL-SG. For clarification, glucose ∩ Sigmacell)=4 represents the intersection of the two sets, while glucose/(Sigmacell ∪ xylan)=117 represents the proteins uniquely associated with growth on glucose as compared to Sigmacell. Other results are interpreted in a similar manner.

While the present invention is susceptible to various modifications and alternative forms, exemplary embodiments thereof are shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description of exemplary embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

DESCRIPTION OF EXEMPLARY EMBODIMENTS In General

The present invention comprises many embodiments. In one embodiment, the invention is a method of digesting a lignocellulosic material, comprising the step of exposing the material to an effective amount of Streptomyces sp. ActE secretome preparation such that at least partial lignocellulosic digestion occurs. In one embodiment of that method, the preparation is a supernatant preparation obtained from a Streptomyces sp. ActE culture. In another embodiment of that method, the preparation is obtained from Streptomyces sp. ActE grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass, and chitin. In another embodiment of that method, the lignocellulosic material is selected from the group consisting of materials that comprise at least 75% cellulose, cellulose/hemicelluloses, xylose, biomass and chitin.

In one embodiment, the invention is a purified preparation comprising the Streptomyces sp. ActE secretome. In one embodiment, the preparation is a supernatant preparation obtained from a Streptomyces sp. ActE culture. In another embodiment of the preparation, Streptomyces sp. ActE is grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass, and chitin.

In one embodiment, the invention is a composition useful for digesting lignocellulosic material comprising one gene or expression product thereof selected from the group consisting of SActE_0237 (GH6) (SEQ ID NO:1), SActE_0236 GH48) (SEQ ID NO:2), SActE_3159 (CBM33) (SEQ ID NO:3), SActE_0482 (GH5) (SEQ ID NO:4), SActE_0265 (GH10) (SEQ ID NO:5), and SActE_2347 (GH5) (SEQ ID NO:6) genes or expression products thereof. In one embodiment, the composition additionally comprises at least one member selected from the group consisting of SActE_0357 (CE4) (SEQ ID NO:7), SActE_0358 (GH11) (SEQ ID NO:8), SActE_1310 (PL3) (SEQ ID NO:9), SActE_3717 (GH9) (SEQ ID NO:10), SActE_4638 (SEQ ID NO:11), SActE_4738 (GH16) (SEQ ID NO:12), SActE_4755 (GH64) (SEQ ID NO:13), SActE_5457 (GH46) (SEQ ID NO:14), SActE_5647 (GH87) (SEQ ID NO:15), and SActE_5978 (PL1) (SEQ ID NO:16) genes or expression products derived thereof.

In one embodiment, the invention is a composition useful for cellulose degradation comprising SActE_0236 (GH48) (SEQ ID NO:2), SActE_3159 (CBM33) (SEQ ID NO:3), SActE_0482 (GH5) (SEQ ID NO:4) and SActE_0237 (GH6) (SEQ ID NO:1) genes or expression product thereof. In one embodiment, the composition additionally comprises at least one member selected from the group consisting of SActE_0357 (CE4) (SEQ ID NO:7), SActE_0358 (GH11) (SEQ ID NO:8), SActE_1310 (PL3) (SEQ ID NO:9), SActE_3717 (GH9) (SEQ ID NO:10), SActE_4638 (SEQ ID NO:11), SActE_4738 (GH16) (SEQ ID NO:12), SActE_4755 (GH64) (SEQ ID NO:13), SActE_5457 (GH46) (SEQ ID NO:14), SActE_5647 (GH87) (SEQ IDNO:15), and SActE_5978 (PL1) (SEQ ID NO:16) genes or expression products derived thereof.

In one embodiment, the invention is a method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of any combinations of genes or expression products derived thereof as disclosed above, wherein the exposed material is at least partially digested.

In one embodiment, the invention is a purified preparation of Streptomyces sp. ActE, wherein the Streptomyces sp. ActE has been grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass and chitin.

In one embodiment, the invention is a purified preparation of Streptomyces sp. ActE, wherein the Streptomyces sp. ActE has been grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon in the substrate is derived from pretreated lignocellulosic material. In one embodiment of the preparation, the pretreated material has been exposed to pretreatment selected from the group consisting of acid hydrolysis, steam explosion, ammonia fiber expansion (AFEX), organosolve, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), ionic liquids (IL), metal-catalyzed hydrogen peroxide treatment, alkaline wet oxidation and ozone pretreatment. In another embodiment of the preparation, the pretreated material is wood.

Specific Embodiments

Applicants have been interested in insects that utilize plant biomass and their associated microbial and fungal communities. Sirex noctilio, a wood boring wasp, is found in pine forests throughout Eurasia and North Africa and is spreading throughout North America and elsewhere (Bergeron et al., 2011). Although the destructive nature of the Sirex infestation is generally considered to arise from a symbiotic relationship between S. noctilio and Amylostereum areolatum, a white rot basidiomycete (Kukor and Martin, 1983; Klepzig et al., 2009; Bergeron et al., 2011), the role of cellulolytic microbes has not been previously considered in the context of the infestation or symbiosis. Streptomyces sp. SirexAA-E [Streptomyces sp. ActE, also referred to herein as “ActE” (Adams et al., ISME J. 5:1321-1231, 2011)], was isolated from the ovipositor mycangium of S. noctilio (Adams et al., 2011). Applicants hypothesized that ActE is inoculated into insect feeding tunnels upon infestation along with the symbiotic fungus. Thus, Applicants were interested to learn how ActE might contribute to the Sirex community.

The present invention will be more fully understood upon consideration of the following non-limiting Examples. All papers and patents disclosed herein are hereby incorporated by reference as if set forth in their entirety.

As used herein, the term “ActE” refers to Streptomyces sp. SirexAA-E, as described in Adams et al., ISME J. 5:1321-1231, 2011. A representative sample of Streptomyces sp. ActE has been deposited according to the Budapest Treaty for the purpose of enabling the present invention. The repository selected for receiving the deposit is the American Type Culture Collection (ATCC) having an address at 10801 University Boulevard, Manassas, Va. USA, Zip Code 20110. The ATCC repository has assigned the patent deposit designation PTA-12245 to the Streptomyces sp. ActE strain.

As used herein, the term “secretome” refers to the plurality of secreted enzymes. For example, ActE secretome refers to the secreted enzymes from Streptomyces sp. SirexAA-E.

As used herein, the term “lignocellulosic material” refers to any material that is composed of cellulose, hemicellulose, and lignin, wherein the carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin.

As used herein, the term “biomass” refers to a renewable energy source, is biological material from living or recently living organisms. As an energy source, biomass can either be used directly, or converted into other energy products such as biofuel. Biomass includes plant or animal matter that can be converted into fibers or other industrial chemicals, including biofuels. Industrial biomass can be grown from numerous types of plants, including miscanthus, switchgrass, hemp, corn, poplar, willow, sorghum, sugarcane, bamboo, and a variety of tree species, ranging from eucalyptus to oil palm (palm oil). Thus, biomass can include wood biomass and non-wood biomass.

The present invention has multiple embodiments. All embodiments are related to Applicants' discovery of improved lignocellulosic digestion and utilization using proteins and genes obtained from the Streptomyces sp. ActE secretome.

ActE Isolates and Secretomes

Streptomyces sp. SirexAA-E may be isolated from ovipositor mycangia of S. noctilio. In Adams, et al, S. noctilio were collected from a population in Pennsylvania, USA. Infested trees were cut and transported to USDA Pest Survey, Detection, and Exclusion Lab in Syracuse, N.Y., USA (Zylstra et al. (2010) Agric. Forest. Entomol. in press). Four adult females and six larvae from the Pennsylvania population were sampled, and cultures of bacteria derived from these insect samples were screened for cellulose degradation.

Prior to sampling for bacteria, all insects were typically surface sterilized in 95% ethanol for 1 minute and then rinsed twice in sterile phosphate-buffered solution (1×PBS). Larval guts and adult ovipositors and mycangia were removed surgically. These segments and the body were ground separately in 1 ml 1×PBS using a sterilized mortar and pestle. 50 μl of three 100-fold dilutions of each insect part were plated onto yeast and malt extract agar (Becton, Dickinson and Company, Sparks, Md., USA), acidified yeast malt extract agar (for gut dissections only), 10% tryptic soy agar (Becton, Dickinson and Company, Sparks, Md., USA), and agar supplemented with chitin (MP Biomedicals, Solon, Ohio). Petri dishes were stored at room temperature in darkness for at least three days until visible colonies formed, except for Petri dishes with chitin agar that were stored for at least one month.

All isolates were typically screened for production of cellulolytic enzymes on carboxymethyl cellulose (CMC) (Teather R M, Wood P J (1982); incorporated herein by reference as if set forth in its entirety). Isolates that tested positive on CMC were then studied further. Assays on CMC, AFEX-treated corn stover at three pH levels, and microcrystalline cellulose were typically performed to assess growth and degradation ability of each insect-derived bacterial isolate. Isolates capable of degrading CMC were further analyzed genomically to identify isolates with high CAZy content relative to one another and relative to known organisms. Streptomyces sp. ActE was selected based on its CMC degradation and CAZy gene profile.

In one embodiment, secretomes from ActE would be used alone in a first reaction to convert biomass into a hydrolyzed solution of sugars that would be used in a second reaction with a fermentation organism to convert the sugars into usable biofuels. The first and second reaction could occur simultaneously.

In a second embodiment, secretomes from ActE would be combined with secretomes from other organisms, or with enzymes or enzyme compositions, such as Spezyme CP, to increase the activity of both preparations by synergy of the enzymes contained in each preparation.

Preferably, the ActE secretomes would be prepared as supernatants from ActE preparations.

In one embodiment, the supernatant is prepared by centrifugation of the ActE culture for 10 min at 3,000×g, which will pellet the remaining insoluble polysaccharide and adhered ActE cells. The supernatant fraction is filter-sterilized, preferably using a 0.22 μm filter, in order to remove any remaining cells. The supernatant is concentrated, preferably using a 3 kDa cut-off ultrafiltration membrane. The concentration of total protein is determined by Bradford assay (Bradford, 1976). In one preferred embodiment, the proteomic composition of the ActE secretome is that described in FIG. 3 or FIG. 18.

The secretomes obtained from growth on specific lignocellulosic materials, such as cellulose, xylan, cellulosic hemi-cellulosic biomass, and chitin, will have distinct compositions of individual enzymes and also distinct reactivity with different polysaccharides. The cellulosic hemi-cellulosic biomass may be non-wood biomass or wood biomass. For example, the secretome prepared from ActE grown on cellulose has unique enzymes and enhanced reactivity with cellulose and mannan. Also, the secretome prepared from ActE grown on xylan possesses high xylan degradation activity, whereas the secretome from ActE grown on chitin possesses uniquely high chitin degradation activity. Example A discloses the specific secretomes.

When ActE is grown on switchgrass, AFEX-pretreated switchgrass or ionic liquid pretreated switchgrass, the secretome has a protein composition that partially matches that obtained from growth on either cellulose or xylan. However, switchgrass, AFEX-pretreated switchgrass or ionic liquid pretreated switchgrass elicit the appearance of new proteins in the secretome that enhance the degradative ability of the secretome for the plant biomass materials. Applicants envision that the present invention would also apply to other pretreatment methods comprising acid hydrolysis, steam explosion, organosolve, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), metal-catalyzed hydrogen peroxide treatment, alkaline wet oxidation and ozone pretreatment.

The inventors' preliminary data shows synergistic filter paper degrading activity between the ActE secretome and other cellulases from a different organism. Also, addition of a beta-glucosidase to the secretome helps to break down the oligosaccharides (e.g., cellotetraose, cellotriose and cellobiose) released from filter paper into simpler sugars.

Preferably, the secretome would be prepared as a concentrated solution by ultrafiltration. The concentrated material would be mixed with the substrate at weight percentages varying from 0.1% to 20% w/w, with the remainder of the solution containing a buffer substance that controls pH. Trace metals would be added to the reaction. The material would be incubated at the appropriate temperature to allow the reaction to occur, with mixing of the reaction materials. The sample might be equilibrated with air or O₂ gas throughout the reaction time period.

The secretome obtained from growth of ActE on cellulose provides all necessary enzymes for most efficient breakdown of cellulose to cellobiose and mannan to mannose. Weak reaction is observed for breakdown of xylan to xylose and a mixture of mannobiose and mannose.

The secretome obtained from growth of ActE on xylan provides all necessary enzymes for most efficient breakdown of xylan to xylobiose and xylose. Weak reaction is observed for breakdown of cellulose to cellobiose and for breakdown of mannan to mannose.

The secretome obtained from growth of ActE on chitin provides all necessary enzymes for most efficient breakdown of chitin to N-acetylglucosamine. Weak reaction is observed for breakdown of xylan to xylose. Weak reaction is observed for breakdown of cellulose to cellobiose and for breakdown of mannan to mannose.

The secretome obtained from growth of ActE on switchgrass biomass provides all of the necessary enzymes for breakdown of cellulose, xylan, and mannan contained in switchgrass to the constituent monosaccharides and disaccharides. Growth of ActE on switchgrass exposed to different chemical pretreatments changes the composition of enzymes present, which alters the rate of production and yield of the constituent monosaccharides and disaccharides.

The secretome obtained from growth of ActE on cellulose provides the necessary enzymes for breakdown of cellulose to cellobiose. ActE uses cellobiose as the growth substrate, so no enzymes are present to convert cellobiose to glucose.

In order to obtain glucose, a cellobiase or beta-glucosidase would be added. This is a standard practice in biofuels enzymology.

In order to convert cellobiose to glucose, a cellobiase or beta-glucosidase would be added. Addition of cellulases from other organisms can improve the rate of hydrolysis of cellulose, e.g., addition of CelLcc_CBM3a, an engineered enzyme from C. thermocellum covered in Fox and Elsen Patent Application No.: PCT/US2010/037094.

The secretome obtained from growth of ActE on cellulose provides all of the necessary enzymes for breakdown of cellulose to cellobiose in a soluble form. One skilled in the art might purify these proteins directly from the secretome without use of tags or recombinant approaches.

As previously noted, the dominance of cellobiose as a product of cellulose deconstruction by ActE might help to channel cellulolytic activity to only a subset of the diverse microbes found in the Sirex community. Exploiting this community interaction, along with establishing control of the highly regulated patterns of gene expression observed in ActE provides the basis for a new biotechnological route for lignocellulosic digestion. For example, use of ActE secretomes to produce cellobiose will restrict the use of cellulose as a fermentation substrate to only those organisms capable of cellobiose uptake followed by intracellular conversion to glucose and subsequent glycolytic pathway intermediates. This might be achieved by coupling ActE enzymes with a yeast fermentation strain engineered to contain a specific cellobiose transporter and an intracellular cellobiose phosphorylase, leading to the intracellular production of glucose and glucose-1-phosphate.

ActE secretomes can be mixed with cellulosic biomass to convert it to cellobiose and xylose, as in the biofuels industry. For example, one might (1) mix the secretome with paper waste to convert it to a mixture of readily fermentable oligo-, di-, and monosaccharides; (2) mix with animal feeds to increase the digestibility of the biomass to promote animal growth; (3) mix with cotton-based textiles for smoothing or other refinements; (4) mix with waste from the shrimp industry to process solid chitin to soluble constituents; (5) mix with mannan-enriched materials to convert them to mannose and mannobiose. One would also find the secretome useful for commercial food processing or treatment of cellulosic bezoar found in the human stomach.

One embodiment of the present invention is an isolation or purified preparation of Streptomyces sp. ActE.

An isolation of ActE was originally reported by Adams et al., (2011) ISME j doi:10.1038/ismej.2011.14, where it was stated that “Sirex noctilio were collected from infested scots pine, Pinus sylvestris L, in Onondaga County, N.Y., USA in 2008”, and “Microbial isolates were obtained from four adult females and six larvae collected in 2008, and were screened for cellulase activity.” These isolates were screened for cellulolytic ability by growing them on CMC, AFEX-treated corn stover, and microcrystalline cellulose.

Applicants envision that one would wish to prepare ActE isolates on specific nutrient sources for optimization for particular digestion profiles. Therefore, one may wish to prepare ActE on substrates wherein at least 40%, preferably 85% of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass, and chitin.

In a preferred embodiment, ActE would be grown aerobically to maximize the secretion of enzymes that include both oxidative and hydrolytic enzymes capable of the rapid deconstruction of biomass. Since ActE cannot utilize mannose for growth, but efficiently liberates mannose from biomass, mannose would become available for growth of the inoculum of a fermentation organism in co-culture. The likely fact that ActE produces at least one antibiotic that would help maintain culture sterility is another possible advantage to establishment of an effective co-culture.

The high capacity for mannan hydrolysis coupled with the inability of ActE to use mannose as a growth substrate offers unique potential opportunity for expansion of deconstruction enzymology to the use of woody substrates. The deconstruction of woody substrates is considered to be more challenging for biofuels production despite the fact that woody substrates are also considerably more highly enriched in mannan than grass substrates. This unique potential opportunity will be enhanced by ongoing plant engineering research efforts to redefine the proportion of xylan and mannan in plant hemicellulose. The availability of plant material enriched in mannan will be coupled to vigorous conversion to mannose by ActE secretomes, providing a targeted, simply fermented C6 sugar for exclusive use by the fermentation organism.

When sufficient titer of enzymes and fermentation organism have been achieved, facilitated by the vigorous, obligate aerobic growth of ActE and corresponding deconstruction of biomass, the fermentation could be initiated by removal of the air source from the culture vessel. In the anoxic conditions, ActE would cease to grow, and perhaps even lyse to become a protein source for the fermentation organism, which will continue to grow on biomass that is simultaneously being deconstructed by the loading of highly active hydrolytic enzymes originally produced by ActE during the aerobic growth phase.

Applicants envision adding an ActE isolate directly to biomass slurry. More preferably an ActE isolate would be added to the pretreated biomasses in the enzyme hydrolysis step, because ActE is able to grow at wide range of pH. ActE can be genetically modified so that the proteolysis proof secretome will be achieved. Growth on switchgrass elicits the appearance of new proteins in the secretome that enhance the degradative ability of the secretome for the plant biomass materials. Applicants envision that the present invention would apply to the biomasses pretreated by many pretreatment methods comprising AFEX, ionic liquid pretreated, acid hydrolysis, steam explosion, organosolve, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), metal-catalyzed hydrogen peroxide, alkaline wet oxidation and ozone pretreatment.

In one preferred embodiment of the present invention, at least one key enzyme in the secretome can be overexpressed by genetic modification of the ActE strain. Table 1 provides various combinations of genes that can be overexpressed. For example, one may wish to overexpress core cellulose deconstructing enzymes, SACTE_0237, SACTE_0482, SACTE_0236, or SACTE_3159 together with one or more of SACTE_2347, and SACTE_0265. One may wish to overexpress core xylan deconstructing enzymes, SACTE_0265, SACTE_0358, SACTE_0357, SACTE_5978, and SACTE_5230. One may wish to overexpress core mannan deconstruction enzymes, such as SACTE_2347. Additionally, SACTE_4755 and SACTE_4738 may be overexpressed for beta-1,3-glucan deconstruction. One may also overexpress all or some of the aforementioned genes for efficient biomass deconstruction.

In another embodiment of the present invention, at least one key enzyme in the secretome can be overexpressed and secreted by genetic modification of a different microbial host such as Streptomyces lividans, which is used for industrial secretion of proteins (Anne and Van Mellaert. (1993)), or T. reesei, which is used for secretion of enzymes in the biofuels industry (Saloheimo and Pakula, Microbiology, Epub date 2011 Nov. 5).

In another embodiment of the present invention, at least one key enzyme in the secretome can be overexpressed by genetic modification of a different microbial host such as S. cerevisiae or E. coli such that the expressed protein will be retained inside of the host cell. The host cells would then be harvested and used as a delivery agent without need for purification of the entrained enzyme, as described in (Wood et al., 1997. This version of the invention may be useful in the enzymatic pretreatment of agricultural crop materials for consumption by ruminant animals.

Combinations of ActE Genes and Expression Products

Selected minimal genes in each subset were chosen based on the combination of genomic, transcriptomic and secretomic results (See Examples and Table 1). For example, in the cellulose minimal gene set, expression of these genes was relatively enriched in cellulose grown cells, compared to glucose grown cells, also corresponding proteins were highly secreted in response to the cellulose in culture medium. Elected minimal genes were annotated to have cellulose utilization function. A larger set of genes for cellulose utilization were selected based on the enrichment of gene expression in cellulose-grown cells relative to glucose-grown cells, and a functional annotation supports cellulose utilization of these genes. Additionally, neighborhood genes to these selected genes on genome were included as genes regulated under same promoter. Similarly, both minimal and a large set of genes for xylan, chitin, and biomasses were elected.

In one embodiment, the present invention is a composition useful for digesting lignocellulosic material comprising genes or expression products thereof selected from the group consisting of: (a) SActE_0237 (SEQ ID NO:1), SActE_0236 (SEQ ID NO:2), SActE_3159 (SEQ ID NO:3), SActE_0482 (SEQ ID NO:4), SActE_0265 (SEQ ID NO:5), and SActE_2347 (SEQ ID NO:6), and (b) SActE_0357 (CE4) (SEQ ID NO:7), SActE_0358 (GH11) (SEQ ID NO:8), SActE_1310 (PL3) (SEQ ID NO:9), SActE_3717 (GH9) (SEQ ID NO:10), SActE_4638 (SEQ ID NO:11), SActE_4738 (GH16) (SEQ ID NO:12), SActE_4755 (GH64) (SEQ ID NO:13), SActE_5457 (GH46) (SEQ ID NO:14), SActE_5647 (GH87) (SEQ ID NO:15), and SActE_5978 (PL1) (SEQ ID NO:16). In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In one embodiment, one would use at least one member of (a) to digest a preferred lignocellulosic material.

In another embodiment, one would use at least the first four members [SActE_0237 (SEQ ID NO:1), SActE_0236 (SEQ ID NO:2), SActE_3159 (SEQ ID NO:3), and SActE_0482 (SEQ ID NO:4)] of (a) to digest a preferred lignocellulosic material.

In another embodiment, one would use at least one member of (a) and at least one member from (b), to digest a preferred lignocellulosic material.

In a preferred embodiment, one would use all the members of (a) and (b), to digest a preferred lignocellulosic material.

In other embodiments, the combination of genes or expression products thereof in the present invention is dependent on the specific lignocellulosic material to be digested. In one embodiment, a composition optimized for cellulose utilization may include any combinations of ActE genes and expression products disclosed above with at least one member selected from SActE_0265 (GH10) (SEQ ID NO:5) and SActE_2347 (GH5) (SEQ ID NO:6) genes or expression products thereof.

In another embodiment, a composition optimized for xylan utilization may include any combinations of ActE genes and expression products disclosed above with at least one member selected from SActE_0265 (GH10) (SEQ ID NO:5), SActE_0358 (GH11) (SEQ ID NO:8), SActE_0357 (CE4) (SEQ ID NO:7), SActE_5978 (PL1) (SEQ ID NO:16) and SActE_5230 (xylose isomerase) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In another embodiment, a composition optimized for chitin utilization may include any combinations of ActE genes and expression products disclosed above with at least one member selected from SActE_4571 (GH18), SActE_2313 (CBM33), SActE_4246 (GH18), SActE_3064 (GH19), and SActE_5764 (GH18) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In another embodiment, a composition optimized for biomass utilization may include any combinations of ActE genes and expression products disclosed above with SActE_5457 (GH46) (SEQ ID NO:14) genes or expression products thereof.

In another embodiment, a composition optimized for mannan utilization may include any combinations of ActE genes and expression products disclosed above with SactE_2347 (GH5) (SEQ ID NO:6) genes or expression products thereof.

In another embodiment, a composition optimized for beta-1,3-glucan utilization may include any combinations of ActE genes and expression products disclosed above with at least one member selected from SActE_4755 (GH64) (SEQ ID NO:13) and SActE_4738 (GH16) (SEQ ID NO:12) genes or expression products thereof.

In another embodiment, a composition optimized for pectin release utilization may include any combinations of ActE genes and expression products disclosed above with SActE_1310 (PL3) (SEQ ID NO:9) gene or expression products derived thereof.

In another embodiment, a composition optimized for alginate release utilization may include any combinations of ActE genes and expression products disclosed above with SActE_4638 (SEQ ID NO:11) gene or expression products derived thereof.

In another embodiment, a composition optimized for galactose release utilization may include any combinations of ActE genes and expression products disclosed above with SactE_5647 (GH87) (SEQ ID NO:15) gene or expression products derived thereof.

In another embodiment, the present invention is summarized as a composition useful for xylan degradation comprising SActE_0265 (GH10) (SEQ ID NO:5) and SActE_0358 (GH11) (SEQ ID NO:8) genes or expression products thereof.

In another embodiment, the present invention is summarized as a composition useful for xylan degradation comprising SActE_0265 (GH10) (SEQ ID NO:5), SActE_0358 (GH11) (SEQ ID NO:8), SActE_0265 (GH10) (SEQ ID NO:5), SActE_0358 (GH11) (SEQ ID NO:8), SActE_0357 (CE4) (SEQ ID NO:7), SActE_5978 (PL1) (SEQ ID NO:16), and SActE_5230 (xylose isomerase) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In another embodiment, the present invention is summarized as a composition useful for biomass degradation comprising SActE_0237 (GH6) (SEQ ID NO:1), SActE_0482 (GH5) (SEQ ID NO:4), SActE_3159 (CBM33)(SEQ ID NO:3), SActE_0236 (GH48) (SEQ ID NO:2), SActE_3717 (GH9) (SEQ ID NO:10), SActE_0265 (GH10) (SEQ ID NO:5), SActE_0358 (GH11) (SEQ ID NO:8), SActE_2347 (GH5) (SEQ ID NO:6) and SActE_1310 (PL1) genes or expression products thereof. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In one embodiment, the present invention is a composition useful for digesting lignocellulosic material comprising genes or expression products thereof selected from the group consisting of: (a) SActE_0237 (SEQ ID NO:1), SActE_0236 (SEQ ID NO:2), SActE_3159 (SEQ ID NO:3), SActE_0482 (SEQ ID NO:4), SActE_0265 (SEQ ID NO:5), and SActE_2347 (SEQ ID NO:6) (for cellulose); (b) SActE_0265 (SEQ ID NO:5), SActE_0357 (SEQ ID NO:7), SActE_0358 (SEQ ID NO:8), SActE_5230 and SActE_5978 (for xylan); (c) SActE_2313, SActE_3064, SActE_4246, SActE_4571 and SActE_5764 (for chitin); (d) SActE_2347 (for mannan); and (e) SActE_0236 (SEQ ID NO:2), SActE_0237 (SEQ ID NO:1), SActE_0265 (SEQ ID NO:5), SActE_0358, SActE_1310, SActE_2347 and SActE_3159 (for biomass). In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In one embodiment, one would use at least two members of (a), (b), (c), (d) or (e) to digest a preferred lignocellulosic material.

In another embodiment, one would use at least three members.

In a preferred embodiment, one would use all members of (a), (b), (c), (d) or (e).

In another embodiment, one would add gene expression products from the list in Table 1 to a substrate to be digested. For example, for preferred cellulose digestion, one would select at least two members of (a), as described above, and at least one member of the “additional useful genes” in Table 1.

In the case of cellulose degradation, the inventors believe SACTE_3159, SACTE_0237, SACTE_0482, and SACTE_0236 act cooperatively to create nicks and hydrolyze cellobiose units from crystalline cellulose.

ActE key genes can be transferred into known cellulolytic organisms in order to enhance the cellulolytic ability of these organisms. A cellulolytic fungus, T. reesei, has been studied for industrial applications, and can be genetically modified. Applicants' data support synergism of cellulolytic ability of enzymes from different species. A chromosomal gene transfer can be performed into T. reesei by protoplast transformation with a high copy plasmid carrying one or more of the ActE cellulolytic key genes.

A chromosomal or a non-chromosomal gene transfer can be made into a yeast species such as Saccharomyces cerevisiae. For non-chromosomal gene transfer, a high copy plasmid carrying a cassette of five minimal genes (SACTE_0236, SACTE_0237, SACTE_0482, SACTE_3717 and SACTE_3159) would be used to confer cellulolytic and mannanolytic capability to the yeast strain. Similar approaches could be used to confer xylanolytic and chitinolytic capability using combinations of the genes described herein.

One might wish to recombinantly express the disclosed enzymes in E. coli in order to achieve high yield of each enzyme. As is shown in the synergistic result in Example 18, cellulose degradation can be improved by combination of ActE enzymes to enzymes from other organisms.

FIG. 18 shows Spectra count of proteins identified on each substrate, where top 95% most abundant proteins were highlighted green, light purple, purple, blue, orange, pink, light blue and yellow on glucose, cellobiose, cellulose, xylan, switchgrass, AFEX-SG, IL-SG and chitin, respectively.

Applicants envision that one would use a composition comprising at least one member of the abundant proteins, e.g., those highlighted proteins in FIG. 18, for digesting the corresponding lignocellulosic materials. For example, to digest a cellulose material, one would choose at least one gene or expression products thereof selected from the group consisting of SACTE_0237, SACTE_0236, SACTE_2347, SACTE_3159, SACTE_0482, SACTE_0265, SACTE_0357, SACTE_4439, SACTE_0562, SACTE_0358, SACTE_4343, SACTE_1546, SACTE_1310, SACTE_4638, SACTE_5668, SACTE_3717, SACTE_3590, SACTE_2172, SACTE_4571, SACTE_5978, SACTE_6428, SACTE_2313, and SACTE_0366. In a preferred embodiment, the composition comprises at least three or four of the genes or expression products.

In one preferred embodiment, one would use all the highlighted proteins for digesting the corresponding lignocellulosic materials.

In another embodiment, one would add gene expression products from the list in Table 1 to a substrate to be digested. For example, for preferred cellulose digestion, one would select at least one member of the abundant proteins, as described above, and at least one member of the “additional useful genes” in Table 1.

TABLE 1 ActE genes or expression products useful for lignocellulosic degradation. Gene or Expression Product Combinations Preferred subsets Additional Useful Genes SACTE_0236, SACTE_0237, Cellulose SACTE_0229, SACTE_0230, SACTE_3159, SACTE_0482 degradation SACTE_0231, SACTE_0232, SACTE_2347, and SACTE_0233, SACTE_0234, SACTE_0265. SACTE_0235, SACTE_0480, SACTE_0481, SACTE_0483, SACTE_0562, SACTE_0563, SACTE_0733, SACTE_0734, SACTE_2286, SACTE_2287, SACTE_2288, SACTE_2289, SACTE_3158, SACTE_4737, and SACTE_6428 SACTE_0265, SACTE_0357, Xylan degradation SACTE_0364, SACTE_0365, SACTE_0358, SACTE_5230 SACTE_0366, SACTE_0368, and SACTE_5978 SACTE_0369, SACTE_0370, SACTE_0527, SACTE_0528, SACTE_5227, SACTE_5228, SACTE_5229, SACTE_5858, and SACTE_5859 SACTE_2313, SACTE_3064, Chitin degradation SACTE_0080, SACTE_0081, SACTE_4246, SACTE_4571 SACTE_0844, SACTE_0846, and SACTE_5764 SACTE_0860, SACTE_3063, SACTE_4858, SACTE_6493 and SACTE_6494 SACTE_2347 Mannan degradation SACTE_1310 Pectin degradation SACTE_4638 Alginate release SACTE_5647 Galactose release SACTE_5648 SACTE_4738 and Beta-1,3-glucan SACTE_4737, SACTE_4739 and SACTE_4755 degradation SACTE_4756 SACTE_0236, SACTE_0237, Cellulose and SACTE_3065, SACTE_4730, SACTE_0265, SACTE_0358, hemicelluloses SACTE_4755, and SACTE_5166 SACTE_0482, SACTE_1310, degradation SACTE_2347, SACTE_3159 and SACTE_3717

In one embodiment, the present invention is a method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of enzymes, wherein the exposed material is at least partially digested. The enzymes may be ActE secretomes, and ActE secretomes may be prepared and isolated using the methods described above.

In another embodiment, the composition of enzymes for a method for digesting a lignocellulosic material may include ActE secretomes in a combination with secretomes from other organisms, or with enzymes or enzyme compositions, such as Spezyme CP, to increase the activity of both preparations by synergy of the enzymes contained in each preparation.

In another embodiment, the composition of enzymes for a method for digesting a lignocellulosic material may be any combinations of ActE genes and expression products as described above.

EXAMPLES

Materials and Methods

Genome Analysis. The complete genome sequence of Streptomyces sp. SirexAA-E (ActE, taxonomy ID 862751) was determined by the Joint Genome Institute, project ID 4086644. Gene annotation models were predicted using Prodigal (Hyatt, et al., 2010), examined using Artemis (Rutherford, et al., 2000), and are available at NCBI with the following accession numbers, GenBank: CP002993.1; RefSeq: NC_015953.1. Carbohydrate-active enzymes were annotated by comparison of all translated open-reading frames to the CAZy database (Cantarel, et al., 2009). We collected CAZy annotated genes from the CAZy database (www.cazy.org). We then used BLASTP to compare all ActE protein-coding sequences to the CAZy database and to the pfam database (ftp://ftp.ncbi.nih.gov/pub/mmdb/cdd/little_endian/Pfam_LE.tar.gz). These two annotations were then crosschecked, and proteins annotated by both databases were identified as our final CAZy annotation. Secreted proteins were identified by SignalP, TatP, and SecretomeP analyses. BLAST was used to identify sequence orthologs in other organisms. Secondary metabolite gene clusters were identified by AntiSmash analysis (Medema, et al., 2011). CebR boxes were identified by using BLAST comparison of the S. griseus CebR box sequence to the ActE genome (Marushima, Ohnishi, et al., 2009). Networks of expression and functional categories were visualized using Cytoscape (Shannon, et al., 2003)

Biomass Substrates. Switchgrass and AFEX-treated switchgrass were obtained from Great Lakes Bioenergy Research Center. Extensively washed ionic liquid-treated switchgrass was the generous gift of Dr. Masood Hadi (Joint BioEnergy Institute). Wood kraft pulp preparations were the generous gift of Dr. Xuejun Pan (University of Wisconsin Department of Biosystems Engineering).

The complete genome sequence of Streptomyces sp. SirexAA-E (ActE, taxonomy ID 862751) was determined by the Joint Genome Institute, project ID 4086644. Gene annotation models were predicted using Prodigal (Hyatt, et al., 2010), examined using Artemis (Rutherford, et al., 2000), and are available at NCBI with the following accession numbers, GenBank: CP002993.1; Ref Seq: NC_015953.1. Carbohydrate-active enzymes were annotated by comparison of all translated open-reading frames to the CAZy database (Cantarel, et al., 2009). We collected CAZy annotated genes from the CAZy database (See CAZy's website for detail information). We then used BLASTP to compare all ActE protein-coding sequences to the CAZy database and to the pfam database (the pfam database can be found in the website of the National Institute of Health (NIH)). These two annotations were then crosschecked, and proteins annotated by both databases were identified as our final CAZy annotation. Secreted proteins were identified by SignalP, TatP, and SecretomeP analyses. BLAST was used to identify sequence orthologs in other organisms. Secondary metabolite gene clusters were identified by AntiSmash analysis (Medema, et al., 2011). CebR boxes were identified by using BLAST comparison of the S. griseus CebR box sequence to the ActE genome (Marushima, Ohnishi, et al., 2009). Networks of expression and functional categories were visualized using Cytoscape (Shannon, et al., 2003)

RNA microarray. ActE was grown in minimal medium plus the indicated substrate for 7 days. The cell pellet was separated from the culture medium by centrifugation for 10 min at 3000×g. Microarray experiments were carried out as reported previously (Riederer, et al., 2011). The total RNA was extracted from the cell pellet and purified. The University of Wisconsin Gene Expression Center carried out the syntheses of cDNA and array hybridizations. Four-plex arrays were constructed by Nimblegen and hybridized with 10 μg of labeled cDNA. ArrayStar (v4.02, DNASTAR, Madison, Wis.) was used to quantify and visualize data. All analyses were based on three or more biological replicates per carbon source. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire data set. Unless otherwise specified, expression levels are based on log 2 values and statistical analysis of the datasets were performed using the moderated t-test.

Preparation of Secretomes. Supernatants obtained from different culture media were prepared by centrifugation of the culture medium for 10 min at 3000×g, which removed the remaining insoluble polysaccharide and adhered cells. The supernatant fraction was then passed through a 0.22-μm filter in order to remove any remaining cells. For enzymatic assays, the secretomes were concentrated using a 3-kDa cut off ultrafiltration membrane. The concentration of secretome protein was determined by Bradford assay, and the typical yield was ˜150-300 mg of total secreted protein per liter of culture medium.

Extracellular Protein Profiles. Extracellular proteins from culture secretomes were precipitated with trichloroacetic acid (TCA), resuspended in denaturing sample buffer (SDS and 2-mercaptoethanol), and separated by SDS-PAGE in 4-20% gels. Protein bands of interest were excised from the gel, digested with trypsin, desalted with C18 pipette tips (Millipore, Billerica, Mass.) and identified by MALDI-TOF (MDS SCIEX 4800 MALDI TOF/TOF, Applied Biosystems, Foster City, Calif.). Additional samples from the same culture secretomes were analyzed by LC-MS/MS to identify highly abundant proteins in the sample.

Ion exchange separation of the ActE secretome. The ActE cellulose secretome was diluted with cold deionized water until the ionic strength was less than 50 mS. The diluted sample was loaded onto an AKTApürifier™ chromatography station equipped with a 16/10 MonoQ FF ion exchange column. The column was washed with 100 mL of 10 mM phosphate, pH 6.0, to remove unbound proteins. The bound proteins were eluted in a linear, 200 mL gradient of NaCl from 0 to 0.8 M in the same buffer. Fractions from the gradient elution were collected and separated by SDS PAGE. The proportional contribution of individual proteins in each fraction was estimated from SDS PAGE. Individual protein bands from each fraction were cut from the gel and submitted for LC-MS/MS analysis to confirm their identities.

LC-MS/MS Analyses. These experiments were performed at the University of Wisconsin Biotechnology Center. Samples were prepared by TCA precipitation of 100 ng of total secreted protein from 7-day old culture supernatants. Protein samples were digested with trypsin (sequencing grade trypsin, Promega, Madison, Wis.) and were desalted using C18 pipette tips (Millipore, Billerica, Mass.). High-energy collision dissociation (HCD) MS analyses employing a capillary LC-MS/MS were performed on an electrospray ionization FT/ion-trap mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific, San Jose, Calif.). The MS and MS/MS spectra were searched against the spectra obtained from the ActE proteome by using Scaffold (Scaffold_3_00_06, Proteome Software, Portland, Oreg.).

Enzyme Activity Measurements. Reducing sugar assays were carried out by mixing secretome preparations with polysaccharide-containing substrates including cellulose (either Whatman #1 filter paper or Sigmacell-20 as indicated), xylan, chitin, mannan, switchgrass, AFEX pretreated switchgrass, or ionic-liquid pretreated switchgrass²⁴. After incubation in 0.1 M sodium phosphate, pH 6 at 40° C. for 20 h, the reducing sugar content was detected by dinitrosalicylic acid assay (Miller, 1959) and calibrated by using glucose, xylose, or mannose as standards. Purified polysaccharide preparations had negligible background response in the absence of added enzymes. Cellobionic and gluconic acids were assayed by a coupled enzyme assay (K-GATE system, Megazyme, Bray Ireland). Spezyme CP was obtained from Genencor with batch number #4901522860. The distributions of soluble sugar oligomers obtained from secretome reactions were determined using a Shimadzu Liquid Chromatograph HPLC system (Shimadzu Scientific Instruments, Columbia, Md.) equipped with a refractive index detector (RID-10A) and a Phenomenex Rezex RPM-monosaccharide column. The temperature was maintained at 85° C. and Milli-Q water was used as the mobile phase at 0.6 mL min⁻¹ flow rate. Glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose (Sigma) were used as standards. The integrated areas of peaks were analyzed by EZ start 7.2 SP1 software (Shimadzu).

Fractions obtained from the ion exchange separation of the ActE cellulose secretome were combined as unary, binary, ternary, and quaternary assemblies where the total protein concentration was fixed and the individual fractions contributed all, halves, thirds, or quarters of the total protein. The most active fraction was assembled from a ternary combination of fractions containing the following enzymes: fraction 1, SACTE_3159 (CBM33/CBM2 oxidative endocellulase, 95%) and SACTE_4738 (GH16 β-1,3 endoglucanase, 5%); fraction 2, SACTE_0237 (GH6 exocellulase, 60%), SACTE_0482 (GH5 endocellulase, 25%), SACTE_0237 (β-1,3 glucanase, 10%) and SACTE_3159 (oxidative endocellulase, <5%); and fraction 3, SACTE_0236 (GH48 exocellulase, 75%), SACTE_3717 (GH9 endocellulase, 20%) and SACTE_5457 (GH46 chitinase, 5%).

Cellobionic and gluconic acids were assayed by a coupled enzyme assay (K-GATE system, Megazyme, Bray Ireland), either with or without the addition of a large excess of β-glucosidase (Cat. No. 31571, Lucigen, Middleton, Wis.).

Two lots of Spezyme CP were obtained from Genencor (#4900901244, Jan. 27, 2010 and #4901522860, Sep. 2, 2011). The specific activity of these two preparations was indistinguishable.

HPLC Analysis. The distributions of soluble sugar oligomers obtained from secretome reactions without and with the addition of excess β-glucosidase (Lucigen) were determined using a Shimadzu Liquid Chromatograph HPLC system (Shimadzu Scientific Instruments, Columbia, Md.) equipped with a refractive index detector (RID-10A) and a Phenomenex Rezex RPM-monosaccharide column. The temperature was maintained at 85° C. and milli-Q water was used as the mobile phase at 0.6 mL min⁻¹ flow rate. Glucose, cellobiose, cellotriose, cellotetraose, and cellopentaose (Sigma) were used as standards. The integrated areas of peaks were analyzed by EZ start 7.2 SP1 software (Shimadzu).

For the experiments shown in FIG. 21, the ActE secretome (1 μg total protein); CelLcc_CBM3a (1 μg); ActE secretome (0.5 μg) and CelLcc_CBM3a (0.5 μg); or Spezyme CP (1 μg total protein) were used. The products of the enzyme reactions detected by HPLC were: ActE secretome, 95% cellobiose, 5% glucose; CelLcc_CBM3a reaction, 90% cellobiose, 10% glucose; ActE & CelLcc_CBM3a, 5% cellotetraose, 80% cellotriose, 15% cellobiose; Spezyme CP, 33% cellobiose, 67% glucose. All products could be converted to glucose in the presence of excess β-glucosidase.

CelLcc_CBM3a. The nucleotide and amino acid sequence of CelLcc_CBM3a is shown in FIG. 22. CelRcc_CBM3a is an engineered exoglucanase composed of the catalytic core of C. thermocellum CelL (Cthe_0405, residues 32 to 429) fused to a C. thermocellum-derived linker sequence and the CBM3a domain from Cthe_3077, the CipA scaffoldin. This construct was created to better understand the performance of enzymes that are normally targeted to the clostridial cellulosome. The replacement of the dockerin domain in Cthe_0405 with the CBM3a domain abrogates the need for a cellulosomal attachment to obtain maximal catalytic activity from CelLcc_CBM3a on solid substrates. The indicated nucleotide sequence was sub-cloned into wheat germ cell-free translation (Makino et al., 2010) and E. coli expression vectors (Blommel et al., 2009) for protein production. CelLcc_CBM3a was purified by standard immobilized metal (Ni²⁺) chromatography. There was no difference in the specific activity of the protein prepared by these two methods.

Example 1 ActE Exhibits High Cellulolytic Activity Relative to other Cellulolytic Organisms

Prokaryotes such as Streptomyces are often easier to grow than eukaryotes (i.e., fungi such as T. reesei), and aerobes are often easier and more energetically efficient to grow than anaerobes. Streptomyces may also have an advantage of producing antibiotics that limit the ability of other organisms to contaminate the culture medium during growth (Galm et al., 2011; Susi et al., 2011). This may be of advantage during large-scale culture with non-sterile biomass materials such as will be encountered in the biofuels industry.

When compared to other cellulolytic organisms (FIG. 1 and FIG. 6), ActE grows well on pure cellulose substances including amorphous cellulose (cellulose treated with phosphoric acid so as to remove all crystalline structure), filter paper (containing a mixture of amorphous and crystalline cellulose) and Sigmacell (primarily in the crystalline state as determined by X-ray powder diffraction), as well as other polysaccharides such as beta-1,3-glucan (callose), xylan, and chitin. ActE also grows well on biomass samples such as corn stover, ammonia-fiber expansion pretreated corn stover, switchgrass, ammonia-fiber expansion pretreated switchgrass, ionic liquids pretreated switchgrass, bleached spruce wood kraft pulp, and unbleached lodgepole pine kraft pulp.

FIG. 1 compares the ability of ActE, S. coelicolor A3(2) (NCBI taxonomy ID 100226) and S. griseus (NCBI CP002993.1; RefSeq: NC_015953.1) to grow in minimal medium containing filter paper as the only carbon and energy source. These images demonstrate the considerably different capabilities of the three ostensibly cellulolytic organisms. Thus ActE completely destroys the filter paper and achieves high cell density, while the two other, reputedly highly cellulolytic strains are only capable of weak colony formation attached to the filter paper. This result establishes that ActE has uniquely high cellulolytic capacity relative to other Streptomyces strains reported to also have this capability (Forsberg et al., 2011). In fact, the images of FIG. 1 and FIG. 6 demonstrate ActE has cellulolytic capacity rivaling that of T. reesei strain Rut-C30, which is widely acknowledged to be the industrial benchmark for cellulolytic capacity (Merino and Cherry, Adv. Biochem. Eng. Biotechnol. 108:95-120, 2007).

Example 2 Pretreatments Useful for Generating Fermentable Sugars

In the biofuels arena, the desired cellulose fractions of plant biomass are protected by the crystalline packing of the individual cellulose strands, and by the surrounding coating of hemicellulose and lignin. In order to most efficiently access the cellulose, chemical pretreatments are required to “loosen up” the plant cell wall structure. In this context, “loosen up” may mean removal of the lignin fraction, partial hydrolysis of feruloyl and acetyl esters present in hemicellulose, and changes in the crystallinity of the cellulose. An optimal pretreatment retains all fractions of biomass (i.e., lignin, hemicellulose and cellulose) in physical states that can be subsequently used by microbes and enzymes as substrates.

Ammonia-fiber expansion is a pretreatment that uses a combination of ammonia gas, low pressure, and low temperature to effect the loosening process (Balan et al., 2009; Chundawat et al., 2011; International Patent Publication No.: WO 2010/125679). It is particularly effective with grasses, and retains all fractions of the biomass for subsequent valorization without introducing water or salts into the biomass. Ionic liquids pretreatment comprises mixing a charged chemical substance (i.e., the ionic liquid) in equal mass proportions with the biomass material. Interactions between the ionic liquid substance and the biomass cause the crystalline structure of cellulose to convert to an amorphous state (Cheng et al., 2011; Li et al., 2011) but the biomass also becomes heavily contaminated with the ionic liquid during this pretreatment, requiring extensive washing with water, a valuable resource in many localities. Kraft pulping is a method for production of paper from wood that involves treatment of the biomass material with strong alkali, sodium sulfite and moderate temperature, resulting in destruction of the lignin and hemicellulose from the desired cellulose fraction; the final biomass material is also heavily contaminated with salts that also requires extensive washing with water to remove. Acid pretreatments retain the lignin and cellulose but destroy the hemicellulose fraction, and in doing so create toxic substances derived from the decomposition of hemicellulose. Because of the need to neutralize the acid, this pretreatment generates a large contamination of salt that also requires extensive washing with water. SPORL is an acidic pretreatment that uses sulfuric acid, elevated temperature, and sodium bisulfite to effect the pretreatment (Wang et al., 2009; Tian et al., 2011). In SPORL, the lignin and hemicellulose are destroyed and cellulose is recovered, but the cellulose is again heavily contaminated with salts and toxic substances derived from chemical decomposition of hemicellulose.

ActE secretomes are highly effective for degradation of lignocellulosic material pre-treated with AFEX. ActE secretomes are also effective for degrading lignocellulosic material pretreated with ionic liquids, Kraft pulping, acid or SPORL and for degrading untreated lignocellulosic material.

Example 3 ActE Genome has High Content of Genes Encoding Carbohydrate Active enZymes (CAZy) Relative to other Cellulolytic Organisms

Protein-coding sequences of the ActE genome (Hyatt et al., 2010) were analyzed by BLAST comparison (Altschul et al., 1990) to the Carbohydrate Active enZyme (CAZy) database (Cantarel et al., 2009).

Table 2 compares the genomic characteristics of ActE with well-known soil-isolated Streptomyces that produce antibiotics and with two model cellulolytic bacteria, Clostridium thermocellum and Cellvibrio japonicas (Lynd, Weimer, et al., 2002; Deboy, et al., 2008; Riederer, et al., 2011). Putative biomass-degrading protein-coding sequences from ActE were identified by BLAST analysis of the finished genome to the Carbohydrate Active enZyme (CAZy) database. Among the 6357 predicted protein-coding genes, 167 have one or more domains assigned to CAZy families, including 119 glycoside hydrolases (GHs), 29 carbohydrate esterases (CEs), 6 polysaccharide lyases (PLs) and 85 carbohydrate binding modules (CBMs). ActE contains 45 different types of GH families, 4 PL families, 7 CE families, and 21 CBM families. The number of total CAZy domains and diversity of CAZy families is comparable to other highly cellulolytic organisms.

TABLE 2 Comparison of genomic composition. ActE S. coelicolor S. griseus C. thermocellum C. japonicus Genome size 7414440 8667507 8545929 3843301 4576573 (nt) Proteome size 6357 8153 7136 3173 3750 Total CAZy 167 221 132 103 183 Proteins % CAZy 2.6% 2.7% 1.8% 3.2% 4.9% Proteins^(a) Total GH^(b) 119 154 80 70 124 Total PL^(c) 6 11 4 6 14 Total CE^(d) 29 36 23 20 28 Total CBM^(e) 85 98 68 121 134 antiSMASH 22 24 37 3 4 clusters^(f) Genes in 620 718 1139 89 111 clusters % antiSMASH 9.8% 8.8% 16.0% 2.8% 3.0% ^(a)Proteins classified as Carbohydrate Active Enzymes (CAZy). ^(b)GH, glycoside hydrolase. ^(c)PL, pectate lyase. ^(d)CE, carbohydrate esterase. ^(e)CBM, carbohydrate binding module. ^(f)Putative antibiotic producing gene cluster.

Nearly all publically available Streptomyces genomes encode a relatively high percentage of genes for putative cellulolytic enzymes. Interestingly, ActE and the antibiotic producing Streptomyces, S. griseus and S. coelicolor, shown in Table 2 have similar numbers and compositions of CAZy families, but substantially different genome sizes. However, these antibiotic-producing Streptomyces are not highly cellulolytic (FIG. 1). Relative to S. griseus and S. coelicolor, the ActE genome contains two unique CAZy families but does not possess 16 CAZy families present in these species. However, ActE contains more representatives in 13 CAZy families. Enrichment of certain CAZy families was observed in other highly cellulolytic organisms. For example, C. thermocellum contains 16 genes in the GH9 family alone. It is interesting to consider whether the reduction in total genome size and differences in CAZy composition between ActE and other closely related soil-dwelling Streptomyces might have arisen from evolutionary specialization of ActE, perhaps driven by association with the Sirex-fungal symbiosis.

ActE contained 12 CAZy families not found in the other model cellulolytic organisms shown in FIG. 3, including GHs, CBMs, and PLs. Seven other CAZy categories, primarily hemicellulases, were shared only with T. reesei. ActE had 23 GH, 10 CBM and 2 PL not found in Thermobifida fusca, another cellulolytic Actinomycetales, which had only 1 GH and 1 CBM not found in ActE. The genome sequence revealed C. japonicus (strain Ueda 107) is highly enriched in GH43 enzymes required for hemicellulose utilization, but is missing a key reducing end exocellulase (bacterial GH48) required for robust growth on cellulose [e.g., see page 5459 of (DeBoy et al., 2008)]; both of these enzyme families are present in highly cellulolytic ActE. Furthermore, ActE also contained 6 genes from the CBM33 family, recently shown to catalyze oxidative cleavage of chitin (Vaaje-Kolstad et al., 2010) and cellulose (Forsberg et al. 2011). Thus, ActE has genomic composition overlapping other cellulolytic organisms, but with notable expansion in the CAZy composition for both hydrolytic and oxidative enzymes and the presence of the complete set of enzymes required for efficient cellulose deconstruction.

Example 4 Genome-wide Gene Expression Analysis of ActE CAZy Gene

Gene expression profiles were determined for ActE grown on purified polysaccharides and plant biomass by whole genome microarrays (FIGS. 4 and 5, FIGS. 9 to 14). Genome-wide gene expression was analyzed as a functional annotation network composed of ActE genes (circles) connected to predicted functional groups (triangles; KEGG or CAZy). In FIG. 4, the network was annotated with genome-wide microarray expression data to indicate genes that were differentially expressed when ActE was grown on either AFEX-SG or glucose, and further annotated to indicate normalized expression levels observed during growth on AFEX-SG. While many aspects of metabolism are modestly changed in response to these different carbon sources, the CAZy and ABC transporter categories were substantially enriched in differentially expressed genes (FIG. 4, green circles). Furthermore, pentose sugar metabolism, sulfur metabolism, and some amino acid biosynthesis pathways (e.g., aromatic amino acids) were also highly induced during growth on AFEX-SG relative to other carbon sources (FIGS. 9-14). In contrast, ribosomal, secondary metabolite, and DNA repair genes showed little change in expression across the conditions examined. Within the CAZy functional group, there was a large induction of genes that contained both a GH domain and a CBM2 domain. Among the 11 genes in the ActE genome that contain a CBM2 domain, 6 were induced greater than 4-fold during growth on AFEX-SG. Furthermore, 9 of the 11 CBM2 containing proteins were identified in the secreted proteome (FIG. 3).

Example 5 ActE CAZy Gene Expression is Dependent on ActE Growth Substrate

Given the large number of differentially expressed CAZy genes identified in the network analysis, Applicants analyzed the expression of this group of genes in cultures grown on different carbon sources (FIG. 5, FIG. 15 and FIG. 16). As with other cellulolytic organisms, there was strong correlation between the content of the secreted proteomes and the most highly expressed genes. Of the 167 ActE genes containing CAZy domains, 68 genes (FIG. 5, group 1) showed distinct increases in expression when grown on different polymeric substrates, 14 genes (FIG. 15, group 2) did not show any appreciable level of expression, and 85 genes (FIG. 16, group 3) showed moderate changes in expression with the different substrates. A significant fraction of these genes contained translocation signals for either the Sec or twin-arginine translocation pathways, and genes encoding structural polypeptides for these translocation pathways were also highly expressed. Besides correlation with secreted proteins, the transcriptomic studies also gave insight into co-regulated gene clusters that potentially encode functional units for utilization of different polysaccharides by ActE. In the following, the 130 genes with normalized expression intensities in the top 2% of all genes are described.

During growth on cellulose, four CAZy genes (SACTE_0236, SACTE_0237, SACTE_3159, and SACTE_0482) showed >15-fold increase in transcript abundance (FIG. 5), and the corresponding proteins were highly enriched in the secreted proteome. None of these four were obviously placed in a gene cluster, and the two most highly expressed genes, SACTE_0236 and SACTE_0237, while adjacent on the chromosome, were transcribed in opposite directions. Nevertheless, these four most highly expressed genes and three others that showed >5-fold increase in transcript abundance (SACTE_3717, SACTE_6428, SACTE_2347, Table 3) were associated with a conserved 14 bp palindromic promoter sequence, TGGGAGCGCTCCCA (the CebR binding element). CebR proteins are Lacl/GalR-like transcriptional regulators shown to provide transcriptional control of gene expression in response to the presence of cellobiose or other small oligosaccharides in S. griseus, S. reticuli, and Thermobifida fusca (Marushima, Ohnishi, et al., 2009; Water and Schrempf, 1996; Deng and Fong, 2010). Likewise, the genes (SACTE_2285 to SACTE_2289) encoding a CebR regulator (SACTE_2285), a GH1 protein (β-glucosidase), a two-protein cellobiose transporter system, and an extracellular solute binding protein were associated with a CebR binding element and were also among the most highly expressed genes during growth on cellulose. These latter five genes have 75% or greater sequence identity with the cellobiose utilization operon identified in S. griseus and S. reticuli (Marushima, Ohnishi, et al., 2009; Schlosser and Schrempf, 1996). There were only 15 genes annotated as hypothetical or domain of unknown function (12%) up-regulated during growth on cellulose, a considerably smaller percentage of these than in the entire genome (27%).

TABLE 3 Analysis of upstream DNA sequence elements in ActE genes upregulated during growth on cellulose. Catalytic Fold Locus domain CBM Annotated function Sequence^(a) Rank^(b) change^(b) SACTE_0236 GH48 CBM2 1,4-beta TGGGAGCGCTC 1 21.7 cellobiohydrolase CCA SACTE_0237 GH6 CBM2 1,4-beta TGGGAGCGCTC 2 17.3 cellobiohydrolase CCA SACTE_3159 CBM33 CBM2 Cellulose-binding TGGGAGCGCTC 3 16.2 domain CCA SACTE_0482 GH5 CBM2 Endo-1,4-beta- TGGGAGCGCTC 4 15.4 glucosidase CCA SACTE_2288 Transport systems TGGGAGCGCTC 5 11.2 inner membrane CCA component SACTE_3717 GH9 CBM2 1,4-beta TGGGAGCGCTC 6 9.7 cellobiohydrolase CCA SACTE_6428 CBM33 Chitin-binding, GGGAGCGCTCC 9 7.9 domain 3 CA SACTE_2347 GH5 CBM2 Beta-mannosidase TGGGAGCGCTC 11 5.0 CCA SACTE_2287 Transport systems TGGGAGCGCTC 15 4.3 inner membrane CCA component SACTE_2289 Family 1 TGGGAGCGCTC 19 3.9 extracellular CCA solute-binding protein SACTE 0352 GCN5-related N- TGGGAGCGCTC 22 3.6 acetyltransferase CCA SACTE_2286 GH1 Glycoside GGGAGCGCTCC 27 3.4 hydrolase 1 CA SACTE_0483 CBM2  Cellulose-binding GGGAGCGCTCC 503 1.6 family protein CA SACTE_0562 GH74 CBM2 Secreted cellulase TGGGAGCGCTC 5759 0.7 (endo) CCA SACTE_2285 Lacl family TGGGAGCGCTC 6229 0.6 transcriptional CCA regulator (CebR) ^(a)Predicted binding sequence element found upstream from gene locus. ^(b)Ranking and fold change in expression intensity detected by microarray for ActE genes when grown on cellulose relative to glucose.

Several characteristics distinguished expression during growth on either xylan or chitin. First, unique sets of genes were induced, as there was only 14% and 10% overlap, respectively, when compared to cellulose. Second, ˜33% of the top 2% of genes expressed during growth on either xylan or chitin were annotated as hypothetical or domain of unknown function, which greatly exceeds the unknown fraction in the cellulose secretome. During growth on xylan, two clusters of genes were up-regulated. One extended from SACTE_0357 to SACTE_0370, encoding proteins from the GH11, GH13, GH42, GH43, GH78, GH87, and CE4 families, a Lacl-like transcriptional regulator, a secreted peptidase, and two sets of inner membrane transporters and associated solute binding proteins. Alternatively, during growth on chitin, three CBM33 proteins were up-regulated (SACTE_0080, SACTE_2313, SACTE_6493), and two of these had an immediately adjacent gene encoding a GH18 (SACTE_6494) or GH19 (SACTE_0081) that was up-regulated.

When ActE was grown on biomass samples, 14 additional CAZy genes were uniquely up regulated, and the corresponding proteins were identified in the proteomic analysis of biomass secretomes (FIGS. 3 and 4). A gene cluster extending from SACTE_5858 to SACTE_5864 was uniquely up regulated during growth on biomass. Among these genes, SACTE_5860 and SACTE_5862 are annotated as a twin-arginine translocation pathway protein and an ABC transporter, respectively, while the rest are annotated either as hypothetical protein or as domain of unknown function.

Eight CAZy genes were >4-fold up-regulated during growth on cellulose, including endoglucanases, reducing and non-reducing end exoglucanases, xylanase and CBM33 proteins (FIG. 5, Table 4). During growth on xylan, eight CAZy genes were elevated >4-fold relative to glucose, including exoglucanase, xylanase, pectate lyase and other hemicellulases (Table 4). Furthermore, chitin-grown cells contained 2 up-regulated genes from CAZy families including chitinase (SACTE_4571) and a CBM33 protein [SACTE_2313, an ortholog of oxidative chitin oxidase from S. marcescens (Vaaje-Kolstad et al., 2010)]. Thus on a genome-wide basis ActE selectively expresses small, distinct sets of CAZy genes during growth on pure polysaccharides, which is distinct from the larger numbers of CAZy genes expressed by T. reesei (Herpoel-Gimbert et al., 2008), C. thermocellum (Raman et al., 2009; Riederer et al., 2011), and T. fusca (Chen and Wilson, 2007).

TABLE 4 Streptomyces sp. ActE genes with >4-fold expression increase during growth on pure polysaccharides. Fold increase CAZy Annotation Sigmacell: glc xylan: glc chitin: glc Sigmacell SACTE_6428 CBM33 Chitin-binding, domain 3 7.06 1.64 1.81 SACTE_3159 CBM33, 2 Cellulose-binding domain, 13.03 1.90 1.29 family II, bacterial type SACTE_0358 GH11, CBM60, 36 Glycoside hydrolase, family 6.28 4.01 2.12 11, active site SACTE_0236 GH48, CBM2, 37 Glycoside hydrolase, 48F 19.00 4.93 3.91 SACTE_0482 GH5, CBM2 Cellulose-binding family 11.84 3.01 2.00 II/chitobiase, carbohydrate- binding domain SACTE_2347 GH5, CE3, CBM2, 37 Cellulose-binding family 4.46 1.17 0.99 II/chitobiase, carbohydrate- binding domain SACTE_0237 GH6, CBM2 1,4-beta cellobiohydrolase 15.33 1.12 0.77 SACTE_3717 GH9, CBM4, 2 Carbohydrate-binding, 8.03 2.61 1.55 CenC-like SACTE_2288 Binding-protein-dependent 11.05 4.76 3.26 transport systems inner membrane component SACTE_0168 Transcription regulator LuxR, 7.55 1.53 1.37 C-terminal SACTE_0169 Glyceraldehyde 3-phosphate 5.01 0.75 1.08 dehydrogenase, active site SACTE_3594 Peptidase S1C, 4.52 3.36 2.70 HrtA/DegP2/Q/S SACTE_5228 Binding-protein-dependent 4.20 4.35 3.24 transport systems inner membrane component Xylan SACTE_4029 CE4 Glycoside 1.07 4.35 2.22 hydrolase/deacetylase, beta/alpha-barrel SACTE_0358 GH11, CBM60, 36 Glycoside hydrolase, family 6.28 4.01 2.12 11, active site SACTE_0382 GH2, CBM42 Galactose-binding domain- 1.79 4.18 2.46 like SACTE_1230 GH23 Lytic transglycosylase-like, 1.29 5.64 3.70 catalytic SACTE_0816 GH31 Glycoside hydrolase, family 1.53 4.51 3.27 31 SACTE_0236 GH48, CBM2, 37 Glycoside hydrolase, 48F 19.00 4.93 3.91 SACTE_1290 GH53, CBM61 Galactose-binding domain- 1.43 4.73 2.40 like SACTE_5978 PL1, CBM35 Galactose-binding domain- 2.00 6.86 2.12 like SACTE_5325 Binding-protein-dependent 1.78 8.26 3.76 transport systems inner membrane component SACTE_6023 Galactose-binding domain- 1.92 7.84 3.34 like SACTE_1834 Alkaline phosphatase D- 1.78 7.73 3.98 related SACTE_6100 Sulfate transporter 2.07 7.45 4.75 SACTE_5361 hypothetical protein 1.77 7.20 3.94 SACTE_5163 Lambda repressor-like, DNA- 1.47 6.89 3.29 binding SACTE_6365 Isocitrate 1.88 6.82 4.01 lyase/phosphorylmutase SACTE_0254 Thiolase-like 2.13 6.76 5.02 SACTE_6478 FAD-dependent pyridine 2.00 6.72 4.46 nucleotide-disulfide oxidoreductase SACTE_3570 hypothetical protein 1.61 6.71 3.72 SACTE_0590 Polyketide 1.55 6.67 4.42 cyclase/dehydrase SACTE_3152 Twin-arginine translocation 1.41 6.60 2.98 pathway, signal sequence SACTE_5285 Bacterial bifunctional 1.71 6.54 3.33 deaminase-reductase, C- terminal SACTE_1383 Glycerophosphoryl diester 1.08 6.50 3.51 phosphodiesterase SACTE_4333 Binding-protein-dependent 1.37 6.46 3.58 transport systems inner membrane component SACTE_3876 hypothetical protein 1.21 6.42 2.73 SACTE_6340 Monooxygenase, FAD- 2.82 6.27 3.69 binding SACTE_4237 hypothetical protein 1.82 6.27 2.91 SACTE_5136 NAD(P)-binding domain 2.20 6.27 2.87 SACTE_6561 hypothetical protein 2.92 6.06 5.65 SACTE_0686 Transcription regulator 0.88 6.04 2.72 AsnC-type SACTE_0817 NUDIX hydrolase, conserved 1.96 6.03 3.19 site SACTE_3004 Type II secretion system F 1.67 6.01 4.18 domain SACTE_1835 DoxX 1.66 5.97 3.30 SACTE_1933 hypothetical protein 0.93 5.96 2.77 SACTE_6290 Glyoxalase/bleomycin 1.86 5.95 4.10 resistance protein/dioxygenase SACTE_5583 hypothetical protein 1.33 5.87 4.56 SACTE_0586 hypothetical protein 1.40 5.81 2.90 SACTE_0046 NADH: flavin 2.48 5.75 4.56 oxidoreductase/NADH oxidase, N-terminal SACTE_1096 Mandelate 1.19 5.73 3.32 racemase/muconate lactonizing enzyme, N- terminal SACTE_2897 hypothetical protein 1.18 5.73 3.81 SACTE_5359 Rhs repeat-associated core 1.30 5.70 2.41 SACTE_0200 hypothetical protein 1.34 5.67 3.64 SACTE_0018 hypothetical protein 1.67 5.63 3.58 SACTE_5542 hypothetical protein 2.03 5.61 3.52 SACTE_3137 hypothetical protein 1.46 5.61 3.91 SACTE_0017 DNA helicase, UvrD/REP 2.32 5.58 4.26 type SACTE_0672 hypothetical protein 1.53 5.54 3.20 SACTE_1393 Urease, beta subunit 2.08 5.53 3.67 SACTE_0064 Transcription regulator PadR 2.17 5.52 3.07 N-terminal-like SACTE_1168 Peptidase S1/S6, 0.98 5.51 3.36 chymotrypsin/Hap SACTE_6371 hypothetical protein 1.37 5.51 3.44 SACTE_4334 Binding-protein-dependent 1.46 5.50 3.35 transport systems inner membrane component SACTE_2457 CDP-glycerol 1.07 5.48 3.79 glycerophosphotransferase SACTE_4734 Binding-protein-dependent 1.21 5.44 3.31 transport systems inner membrane component SACTE_3661 hypothetical protein 1.76 5.44 3.25 SACTE_0036 hypothetical protein 1.75 5.43 2.99 SACTE_6005 Citrate synthase-like, core 1.01 5.38 2.90 SACTE_6562 hypothetical protein 2.34 5.36 3.37 SACTE_1937 Major facilitator superfamily 0.88 5.34 3.02 MFS-1 SACTE_6220 Dodecin flavoprotein 2.13 5.32 5.08 SACTE_0778 FMN-binding split barrel 1.13 5.28 2.72 SACTE_5672 Acyltransferase 3 1.33 5.28 3.09 SACTE_5989 Cysteine-rich domain 1.40 5.24 3.11 SACTE_5296 HTH transcriptional 1.42 5.22 2.96 regulator, MarR SACTE_2021 hypothetical protein 1.44 5.17 2.54 SACTE_1845 Transposase, IS4-like 1.69 5.16 3.30 SACTE_1771 Phage T4-like virus tail tube 1.55 5.10 1.71 gp19 SACTE_2583 hypothetical protein 1.38 5.10 3.11 SACTE_5957 Helix-turn-helix, HxIR type 2.38 5.09 3.95 SACTE_4642 hypothetical protein 1.31 5.08 3.05 SACTE_3695 Aminoglycoside/hydroxyurea 1.41 5.03 3.76 antibiotic resistance kinase SACTE_0079 ATPase-like, ATP-binding 2.21 5.01 2.98 domain SACTE_0727 hypothetical protein 2.54 5.00 3.88 SACTE_0019 hypothetical protein 1.37 5.00 2.40 SACTE_6422 Streptomyces 2.40 4.99 3.57 cyclase/dehydrase SACTE_4348 Bacterial extracellular solute- 1.60 4.97 3.06 binding protein, family 5 SACTE_5318 Forkhead-associated (FHA) 1.50 4.93 2.84 domain SACTE_5413 Urease accessory protein 1.94 4.93 2.52 UreF SACTE_5434 Glutathione S-transferase, 2.41 4.93 2.96 C-terminal-like SACTE_6061 Glyoxalase/bleomycin 1.61 4.92 2.18 resistance protein/dioxygenase SACTE_0025 hypothetical protein 1.58 4.92 4.22 SACTE_5552 Transposase, IS4-like 1.94 4.92 3.26 SACTE_4156 HTH transcriptional 1.57 4.86 2.81 regulator, LysR SACTE_5600 hypothetical protein 1.78 4.83 2.01 SACTE_5331 Conserved hypothetical 1.56 4.82 2.96 protein CHP03086 SACTE_0784 hypothetical protein 1.43 4.80 2.65 SACTE_0045 NAD(P)-binding domain 1.74 4.78 3.35 SACTE_5426 Twin-arginine translocation 0.80 4.77 2.68 pathway, signal sequence SACTE_2654 4Fe—4S ferredoxin, iron- 1.30 4.77 2.68 sulfur binding domain SACTE_2288 Binding-protein-dependent 11.05 4.76 3.26 transport systems inner membrane component SACTE_2324 Membrane insertion protein, 0.91 4.75 2.58 OxaA/YidC, core SACTE_0142 Amidohydrolase 2 1.28 4.71 2.65 SACTE_0787 hypothetical protein 1.66 4.70 2.93 SACTE_5790 hypothetical protein 1.28 4.69 2.83 SACTE_6291 hypothetical protein 1.25 4.68 3.13 SACTE_6499 hypothetical protein 1.66 4.67 3.29 SACTE_6548 Lytic transglycosylase-like, 1.97 4.66 3.20 catalytic SACTE_3087 Major facilitator superfamily 1.30 4.66 3.26 MFS-1 SACTE_5512 hypothetical protein 1.79 4.64 3.48 SACTE_0491 hypothetical protein 2.44 4.63 2.71 SACTE_0312 Thiamine pyrophosphate 2.32 4.60 3.49 enzyme, C-terminal TPP- binding SACTE_6130 hypothetical protein 1.47 4.55 2.64 SACTE_3787 Helix-turn-helix type 3 1.38 4.53 2.73 SACTE_0040 hypothetical protein 1.64 4.52 4.80 SACTE_2461 Macrocin-O- 1.07 4.51 3.00 methyltransferase SACTE_5041 hypothetical protein 1.50 4.49 3.25 SACTE_5540 Transposase, 1.79 4.49 2.99 IS204/IS1001/IS1096/IS1165 SACTE_0776 Protein of unknown function 1.34 4.48 2.52 DUF6, transmembrane SACTE_0785 Bacterial TniB 1.67 4.43 2.93 SACTE_0360 Binding-protein-dependent 1.70 4.43 2.39 transport systems inner membrane component SACTE_3569 Protein of unknown function 1.00 4.42 2.78 DUF1023 SACTE_2986 hypothetical protein 1.62 4.42 2.96 SACTE_4732 Twin-arginine translocation 2.08 4.41 2.72 pathway, signal sequence SACTE_5228 Binding-protein-dependent 4.20 4.35 3.24 transport systems inner membrane component SACTE_0406 Binding-protein-dependent 1.34 4.35 2.52 transport systems inner membrane component SACTE_6516 Binding-protein-dependent 2.24 4.34 3.41 transport systems inner membrane component SACTE_1781 hypothetical protein 1.16 4.34 2.56 SACTE_5936 Radical SAM 1.43 4.33 2.23 SACTE_0819 Protein of unknown function 1.50 4.33 2.83 DUF962 SACTE_4539 NERD 1.42 4.32 3.98 SACTE_0532 Binding-protein-dependent 3.47 4.31 2.42 transport systems inner membrane component SACTE_3300 hypothetical protein 1.68 4.31 2.59 SACTE_6277 hypothetical protein 2.24 4.31 3.11 SACTE_0941 Twin-arginine translocation 1.32 4.30 2.63 pathway, signal sequence SACTE_1115 GntR, C-terminal 1.57 4.29 2.63 SACTE_6105 Fatty acid hydroxylase 1.63 4.29 2.78 SACTE_4407 Spherulation-specific family 4 1.19 4.29 4.15 SACTE_5387 hypothetical protein 1.24 4.27 3.08 SACTE_5053 NmrA-like 1.23 4.27 3.05 SACTE_5562 Amino acid ABC transporter, 1.37 4.26 3.75 permease protein, 3-TM domain, His/Glu/Gln/Arg/opine family SACTE_5522 Galactose-binding domain- 1.82 4.26 2.62 like SACTE_5484 Transcription regulator, 1.45 4.21 3.24 TetR-like, DNA-binding, bacterial/archaeal SACTE_6526 Restriction endonuclease, 2.31 4.20 2.40 type IV-like, Mrr SACTE_4164 hypothetical protein 1.06 4.19 2.48 SACTE_4979 Transcription regulator, 1.20 4.19 2.34 TetR-like, DNA-binding, bacterial/archaeal SACTE_0952 hypothetical protein 1.33 4.18 2.02 SACTE_1785 hypothetical protein 1.25 4.17 1.94 SACTE_3454 hypothetical protein 1.46 4.16 2.32 SACTE_1271 Class II aldolase/adducin, N- 1.77 4.16 2.65 terminal SACTE_1760 hypothetical protein 1.38 4.13 2.07 SACTE_0035 hypothetical protein 1.93 4.13 3.13 SACTE_0247 Protein of unknown function 1.30 4.10 2.77 DUF2241 SACTE_3796 F420-dependent enzyme, 1.43 4.10 3.33 PPOX class, family Rv2061, putative SACTE_4641 hypothetical protein 1.43 4.09 2.60 SACTE_4816 Peptidase S26, conserved 1.17 4.09 2.77 region SACTE_2331 Major facilitator superfamily 1.15 4.08 2.20 MFS-1 SACTE_1666 hypothetical protein 1.44 4.07 2.46 SACTE_5867 Mammalian cell entry, 1.79 4.07 2.92 mce1C SACTE_2705 AMP-binding, conserved site 1.38 4.07 2.75 SACTE_6014 Binding-protein-dependent 0.89 4.07 2.51 transport systems inner membrane component SACTE_2018 Putative DNA binding 1.05 4.06 2.63 domain SACTE_5690 Gluconate transporter 1.00 4.05 2.29 SACTE_3243 hypothetical protein 0.91 4.05 2.23 SACTE_0786 Polynucleotidyl transferase, 1.81 4.03 2.98 ribonuclease H fold SACTE_6450 Rhamnose isomerase 2.72 4.02 2.90 related SACTE_0097 Beta-lactamase-related 1.70 4.02 2.52 SACTE_6341 FMN-binding split barrel, 1.82 4.01 2.45 related SACTE_1483 hypothetical protein 0.82 4.01 2.75 SACTE_0754 Uncharacterised protein 1.21 4.00 2.51 family UPF0060 SACTE_5308 Winged helix-turn-helix 1.33 4.00 1.56 transcription repressor DNA- binding SACTE_5862 ABC transporter, conserved 1.87 4.00 3.05 site Chitin SACTE_2313 CBM33 Chitin-binding, domain 3 1.08 1.24 4.77 SACTE_4571 GH18, CBM57, 2 EF-Hand 1, calcium-binding 0.88 1.37 4.08 site SACTE_5381 hypothetical protein 1.31 3.09 10.06 SACTE_5386 hypothetical protein 0.96 1.59 8.49 SACTE_1949 Peptidase M4, thermolysin 1.30 2.16 7.57 SACTE_6519 Binding-protein-dependent 2.00 3.04 7.36 transport systems inner membrane component SACTE_0243 Protein kinase-like domain 1.68 2.55 6.89 SACTE_6520 ABC transporter, conserved 1.03 1.18 6.25 site SACTE_5384 hypothetical protein 1.16 2.39 5.99 SACTE_6463 hypothetical protein 1.28 2.52 5.85 SACTE_6561 hypothetical protein 2.92 6.06 5.65 SACTE_5383 hypothetical protein 1.06 1.69 5.28 SACTE_6518 hypothetical protein 1.66 1.91 5.21 SACTE_4797 hypothetical protein 2.22 0.34 5.19 SACTE_6170 Domain of unknown function 1.47 3.49 5.12 DUF1996 SACTE_6220 Dodecin flavoprotein 2.13 5.32 5.08 SACTE_0254 Thiolase-like 2.13 6.76 5.02 SACTE_2678 Protein of unknown function 1.40 1.13 5.02 DUF397 SACTE_5968 hypothetical protein 1.58 1.31 4.90 SACTE_4757 Acetyl-coenzyme A 1.64 0.59 4.86 carboxyltransferase, C- terminal SACTE_0040 hypothetical protein 1.64 4.52 4.80 SACTE_6100 Sulfate transporter 2.07 7.45 4.75 SACTE_1833 Twin-arginine translocation 1.64 1.56 4.64 pathway, signal sequence SACTE_5583 hypothetical protein 1.33 5.87 4.56 SACTE_0046 NADH: flavin 2.48 5.75 4.56 oxidoreductase/NADH oxidase, N-terminal SACTE_5398 hypothetical protein 1.45 1.73 4.55 SACTE_6144 Twin-arginine translocation 1.21 1.13 4.52 pathway, signal sequence SACTE_6478 FAD-dependent pyridine 2.00 6.72 4.46 nucleotide-disulfide oxidoreductase SACTE_0590 Polyketide 1.55 6.67 4.42 cyclase/dehydrase SACTE_2112 Homeodomain-like 1.44 1.33 4.40 SACTE_0017 DNA helicase, UvrD/REP 2.32 5.58 4.26 type SACTE_5841 Protein of unknown function, 1.90 3.09 4.24 ATP binding SACTE_0025 hypothetical protein 1.58 4.92 4.22 SACTE_3004 Type II secretion system F 1.67 6.01 4.18 domain SACTE_4407 Spherulation-specific family 4 1.19 4.29 4.15 SACTE_0307 Protein of unknown function 1.13 1.79 4.15 DUF320, Streptomyces species SACTE_6290 Glyoxalase/bleomycin 1.86 5.95 4.10 resistance protein/dioxygenase SACTE_5286 hypothetical protein 1.33 3.34 4.07 SACTE_5953 Protein of unknown function, 1.35 2.11 4.05 ATP binding SACTE_6365 Isocitrate 1.88 6.82 4.01 lyase/phosphorylmutase

Example 6 Composition of ActE Secretome is Dependent on ActE Growth Substrate

To identify secreted proteins, supernatants from ActE cultures grown on glucose, cellobiose, cellulose, xylan, chitin, switchgrass, AFEX-SG, and IL-SG were analyzed by LC-MS/MS (FIG. 3 and FIG. 18). The proteins were sorted into a descending rank according to spectral counts, and sets whose spectral counts summed to 95% of the total protein in each secretome are shown. FIG. 3A summarizes the percentages of CAZy families in the detected proteins. The glucose secretome had a protein concentration of ˜0.03 g/L of culture medium, and among the 136 proteins identified only 3% had a CAZy annotation. Indeed, the majority (>90%) likely originated from cell lysis. In contrast, the polysaccharide secretomes had a protein concentration of ˜0.3 g/L of culture medium, a ˜10-fold increase from the glucose secretome. Pectate lyase (SACTE_1310), chondroitin/alginate lyase (SACTE_4638), an extracellular solute binding protein (SACTE_4343), bacterioferritin (SACTE_1546), and catalase (SACTE_4439) were observed in all polysaccharide secretomes. The first two proteins, SACTE_1310 and SACTE_4638, have signal peptides and are thus secreted as part of the response needed for growth on polysaccharides.

FIG. 3 and FIG. 18 further demonstrate that 22 proteins accounted for 95% of the total spectral counts during growth on cellulose; two-thirds were from CAZy families. The five most abundant proteins, in order and representing ˜85% of the total spectral counts, were reducing and non-reducing exoglucanases (SACTE_0236 and SACTE_0237), a CBM33 polysaccharide monooxygenase (SACTE_3159), an endoglucanase (SACTE_0482), and a β-mannosidase (SACTE_2347). The first four proteins encode a non-redundant set of enzymes that likely provide the essential activities required for utilization of crystalline cellulose (Deboy, et al., 2008). Among the 22 most abundant proteins, there were representatives from 9 different GH families, two CE families, two PL families, and two additional CMB33 proteins. Collectively, these secreted proteins represent ˜20% of the CAZy composition in the ActE genome.

There were substantial differences in the composition of the xylan and chitin secretomes as compared to the cellulose secretome (FIG. 3 and FIG. 18). In the xylan secretome, 92 proteins comprise 95% of the detected spectral counts. Twenty GHs from 18 different CAZy families were included, along with 1 CE4 and 2 PL family proteins. Thus, growth on xylan elicits secretion of representatives from half of the total CAZy families found in the ActE genome. The broad distribution of hemicellulytic enzymes in the xylan secretome contrasts with the considerably less diverse composition of the chitin secretome, which consists of 7 representatives from GH18 (e.g., chitinase, endo beta-N-acetylglucosaminidase), 2 from GH19 (e.g., chitinase, lysozyme), and 1 chitinolytic CBM33 (FIG. 18). While chitinolytic CAZy families account for two-thirds of the proteins secreted during growth on chitin, they represent only ˜6% of the diversity of CAZy families found in the genome. These results document the substantially different substrate-specific responses of ActE during growth on different polysaccharides.

The secretomes isolated from cells grown on switchgrass, AFEX-SG, and IL-SG contained the highly abundant secreted proteins identified in the purified cellulose and xylan experiments and some additional proteins. These additional proteins likely reflect cellular response to the more complex composition of polysaccharides present in the biomass samples. The increased diversity of proteins present in the biomass secretome also increased the efficiency of reaction with plant biomass (FIG. 2C). In total, the biomass secretomes contained 31 different CAZy families that contributed to the total spectral counts (˜70% of the CAZy families present in the ActE genome), thus representing coordinated and extensive use of CAZyme families present in the ActE genome for biomass utilization.

The gene loci of the 117 proteins observed only in the glucose secretome are: SACTE_0494; SACTE_0514; SACTE_0541; SACTE_0548; SACTE_0604; SACTE_0669; SACTE_0687; SACTE_0800; SACTE_0810; SACTE_0899; SACTE_1006; SACTE_1045; SACTE_1068; SACTE_1069; SACTE_1111; SACTE_1201; SACTE_1240; SACTE_1285; SACTE_1328; SACTE_1344; SACTE_1368; SACTE_1419; SACTE_1426; SACTE_1506; SACTE_1522; SACTE_1586; SACTE_1650; SACTE_1861; SACTE_1888; SACTE_1934; SACTE_2036; SACTE_2049; SACTE_2068; SACTE_2238; SACTE_2403; SACTE_2431; SACTE_2468; SACTE_2558; SACTE_2645; SACTE_2729; SACTE_2755; SACTE_2756; SACTE_2801; SACTE_2819; SACTE_3012; SACTE_3037; SACTE_3067; SACTE_3086; SACTE_3088; SACTE_3097; SACTE_3219; SACTE_3327; SACTE_3361; SACTE_3371; SACTE_3385; SACTE_3389; SACTE_3392; SACTE_3414; SACTE_3438; SACTE_3511; SACTE_3604; SACTE_3716; SACTE_3896; SACTE_3948; SACTE_3955; SACTE_3956; SACTE_3960; SACTE_3961; SACTE_3989; SACTE_3995; SACTE_4030; SACTE_4031; SACTE_4038; SACTE_4039; SACTE_4073; SACTE_4081; SACTE_4083; SACTE_4145; SACTE_4191; SACTE_4194; SACTE_4205; SACTE_4224; SACTE_4281; SACTE_4283; SACTE_4376; SACTE_4397; SACTE_4399; SACTE_4415; SACTE_4462; SACTE_4501; SACTE_4550; SACTE_4565; SACTE_4566; SACTE_4567; SACTE_4568; SACTE_4591; SACTE_4610; SACTE_4616; SACTE_4618; SACTE_4652; SACTE_4718; SACTE_4768; SACTE_4791; SACTE_4795; SACTE_4830; SACTE_4860; SACTE_4873; SACTE_4926; SACTE_4959; SACTE_5028; SACTE_5081; SACTE_5192; SACTE_5267; SACTE_5482; SACTE_5519; SACTE_5983; and SACTE_6342.

The gene loci of the 9 proteins observed only in the Sigmacell secretome are: SACTE_0236; SACTE_0482; SACTE_0562; SACTE_2313; SACTE_2347; SACTE_3590; SACTE_3717; SACTE_4571; and SACTE_6428.

The gene loci of the 46 proteins observed only in the xylan secretome are: SACTE_0081; SACTE_0169; SACTE_0365; SACTE_0379; SACTE_0383; SACTE_0464; SACTE_0528; SACTE_0549; SACTE_0634; SACTE_0880; SACTE_1003; SACTE_1130; SACTE_1239; SACTE_1324; SACTE_1325; SACTE_1356; SACTE_1364; SACTE_1367; SACTE_1603; SACTE_1680; SACTE_1858; SACTE_1949; SACTE_2768; SACTE_3064; SACTE_4231; SACTE_4246; SACTE_4363; SACTE_4459; SACTE_4483; SACTE_4515; SACTE_4607; SACTE_4612; SACTE_4624; SACTE_4730; SACTE_4755; SACTE_4858; SACTE_5166; SACTE_5230; SACTE_5231; SACTE_5418; SACTE_5457; SACTE_5630; SACTE_5647; SACTE_5682; SACTE_5751; and SACTE_6439.

In the xylan secretome, five proteins accounted for half of the total secreted protein. These were xylanases (GH10 and GH11, respectively; SACTE_0265, 9.7% and SACTE_0358, 8.1%), extracellular xylose isomerase (SACTE_5230, 12.7%), acetyl xylan esterase (CE4; SACTE_0357, 11.7%), and pectate lyase (PL1, SACTE_5978, 6.6%). Among the remaining 98 proteins, there were numerous GH families. Given the complexity of hemicellulose, which is enriched in xylan but also contains many other sugars and many different bonding linkages between these sugars, it is noted that these additional proteins represent many GH families associated with unique hemicellulolytic activities.

Although not analyzed in FIG. 34, the chitin secretome contained ten proteins from the chitinase GH18 (49% of total protein) and GH19 (21%) families. In addition, the CBM33 protein SACTE_2313, having 50% primary sequence identity with the CBP21 chitin oxygenase from S. marcescens, was also detected (3.9%). Insect molt and fungal hyphae provide abundant chitin, likely accounting for the utility of these enzymes in the natural environment. There were 50 other proteins (63 total) that comprised 95% of the chitin secretome. Relative to the glucose, Sigmacell, and xylan secretomes, the following 15 proteins were observed only in the chitin secretome: SACTE_0746, SACTE_0844, SACTE_0860, SACTE_1702, SACTE_2033, SACTE_2059, SACTE_2062, SACTE_2384, SACTE_3685, SACTE_4468, SACTE_4472, SACTE_4727, SACTE_5330, SACTE_5764, and SACTE_6494

The gene loci of the 19 proteins observed only in the switchgrass secretome are: SACTE_0642; SACTE_1130; SACTE_1250; SACTE_1858; SACTE_2033; SACTE_3012; SACTE_3777; SACTE_4198; SACTE_4571; SACTE_4624; SACTE_4669; SACTE_4676; SACTE_4718; SACTE_4738; SACTE_5220; SACTE_5418; SACTE_5685; SACTE_5751; and SACTE_5880.

The gene loci of the 8 proteins observed only in the IL-SG secretome are: SACTE_0132; SACTE_0880; SACTE_2556; SACTE_4246; SACTE_4515; SACTE_4702; SACTE_5231; and SACTE_5330.

There were no proteins observed only in the AFEX-SG secretome when compared to either the switchgrass or IL-SG secretomes.

Example 7 Minimized Size of ActE Enzymes Increases Specific Activity

When ActE is grown on Sigmacell, AFEX-SG, IL-SG, AFEX-CS, unbleached lodgepole pine kraft pulp (UBLPKP) or bleached spruce wood kraft pulp (BSKP), the characteristic secretome consists of the proteins that permit deconstruction of these substrates into sugars that can be used for growth (FIG. 23). Interestingly, ActE is not capable of growing on lodgepole pine pretreated by SPORL, indicating this pretreatment produces toxins that inhibit the growth of highly cellulolytic microbes. When ActE is grown on cellobiose, which it does readily and rapidly, it produces a secretome that is distinct from those obtained from ActE grown on cellulose, xylan or biomass substrates, demonstrating that ActE has highly specific responses to different polymeric substances that are present in biomass. This behavior is distinct from that observed for T. fusca, another cellulolytic Actinomycete, and from C. thermocellum, where each organism produced similar sets of secreted proteins during growth on either cellulose or cellobiose (Chen and Wilson, 2007; Riederer et al., 2011). This result indicates ActE contains a unique regulatory mechanism for controlling cellulose deconstruction genes that can provide exquisite control of their production under desired circumstances.

For a single enzyme from a secretome, (Segel, Enzyme kinetics: behavior and analysis of rapid equilibrium and steady state enzyme systems. Wiley, New York, 1993) the specific activity (μmol/min/mg) is defined as mol of product formed per unit time (i.e., μmol/min) per unit mass of enzyme (i.e., mg). Specific activity is the parameter that must be used in making comparisons of catalytic properties between enzymes with different molecular masses. If two enzyme isoforms yield the same μmol/min, the isoform with the smaller molecular weight will, by definition, have the higher specific activity. In this application, it is relevant to consider the implications of a 10% or more reduction in the mass of an enzyme required to treat gigatonnes of biomass.

In the cellulose secretome, five proteins contributed ˜85% of the total spectral counts. These were reducing and non-reducing end exoglucanases, endoglucanases, and CBM33 (SACTE_0237, SACTE_0236, SACTE_2347, SACTE_0482 and SACTE_3159); xylanase, another endoglucanase, and another CBM33 were also abundant (SACTE_0265, SACTE_3717 and SACTE_6428). According to the definition provided above, size minimization is a way to achieve the desired increases in specific activity. Interestingly, the set of ActE enzymes described above are on average 10% smaller in mass than their closest orthologs from T. fusca (Chen and Wilson, 2007), suggesting size minimization may have occurred in ActE (Table 5). These enzymes also provide all of the requisite catalytic reactions needed for the deconstruction of crystalline cellulose.

TABLE 5 ActE cellulose secretome proteins and corresponding best match in T. fusca. The single protein SACTE_0237 is the best match to both Cel6A and Cel6B suggesting one protein from ActE might replace two proteins from another organism. ActE T fusca Protein Gene locus CAZy residues MW identity coverage Gene locus name residues MW SACTE_0237 GH6 586 49 80 Tfu_1074 Cel6A 441 45844 SACTE_0237 GH6 586 61062 62 93 Tfu_0620 Cel6B 596 63548 SACTE_0236 GH48 954 100726 57 95 Tfu_1959 Cel48A 984 107127 SACTE_2347 GH8 562 57753 45 23 Tfu_2176 Cel9A 880 95203 SACTE_3159 CBM33 362 37787 42 71 Tfu_1665 E8 438 46808 SACTE_0482 GH5 456 47654 51 97 Tfu_0901 Cel5A 466 49807 SACTE_0265 GH10 458 47683 44 95 Tfu_2923 Xyl10A 491 53185 SACTE_3717 GH9 909 96338 61 82 Tfu_1627 Cel9B 998 107045 SACTE_6428 CBM33 222 24668 62 99 Tfu_1268 E7 222 25372 Average identity, coverage 53 82 Sum ActE 4509 473671 with Cel6A 4920 530391 4509 473671 with Cel6B 5075 548095 Percentage with Cel6A 92% 89% with Cel6A, B 5516 593939 Percentage with Cel6B 89% 86% Percentage with Cel6A, B 82% 80%

Example 8 ActE Secretome Specific Activity is Comparable to that of Spezyme CP™

The enzymatic activities of ActE secretomes were compared with a commercial secretome, Spezyme CP. The enzyme cocktail of Spezyme CP was prepared from T. reesei Rut-C30, thus providing a useful, routinely available reference point for the capabilities of other cellulolytic organisms. HPLC analysis showed that the ActE cellulose secretome released cellobiose as the primary product during reaction with cellulose (FIG. 2A, 95% of products), which is distinct from the higher proportion of glucose produced by the T. reesei secretome. Similarly, the primary products from xylan and mannan were xylobiose and mannobiose, respectively. Upon accounting for total glucose equivalents released, the ActE secretome obtained from growth on pure cellulose had specific activity that was about half of that provided by Spezyme CP (FIG. 2A, inset). Interestingly, the ActE secretome obtained from growth on pure cellulose had higher specific activity for deconstruction of pure mannan than Spezyme CP (FIG.2B). Additionally, the ActE secretome obtained from growth on pure xylan had higher specific activity for reaction with pure xylan than Spezyme CP. Cellulose, xylan, and mannan are all abundant in pinewood, thus accounting for the necessity of each of the major catalytic activities detected.

Anion exchange chromatography was performed to fractionate the ActE secretome obtained from cells grown on cellulose as the sole carbon source. We identified fractions that hydrolyzed pure polysaccharides by biochemical assays (FIG. 7), and confirmed the identity of the protein or proteins contained in these fractions by mass spectrometry (FIG. 17). Where multiple polypeptides were present, the identity of each was confirmed by mass spectrometry to correspond to the indicated gene locus. In several cases, these most likely arise from proteolysis of a single protein found in the secretome. Fractions containing the maximum cellulase activity were highly enriched in SACTE_0236 and SACTE_0237, reducing and non-reducing end cellobiohydrolases from the GH6 and GH48 families, respectively. SACTE_0265 and SACTE_2347 were identified as the major proteins present in fractions associated with xylan and mannan hydrolysis, respectively. A CBM33 polysaccharide monooxygenase (SACTE_3159) was also identified in the ion exchange profile. Moreover, beta-1,3 glucanase activity was identified in fractions that were enriched in SACTE_4755.

When ActE was grown on either ammonia fiber expansion-treated switchgrass (AFEX-SG) (Li, C. et al., 2011) or ionic liquid-treated switchgrass (IL-SG), the secretomes had ˜2-fold increase in specific activity relative to the cellulose secretome and were equivalent to Spezyme CP for reaction with both the AFEX- and IL-treated biomass (FIG. 2C) (Li, C. et al., 2011). The ActE secretomes retained greater than 60% of maximal activity for the hydrolysis of AFEX- and IL-SG from 30 to 55° C. and 35 to 47° C., respectively, which is comparable to recent reports on the temperature profile of secretomes from thermophilic biomass-degrading fungi (Tolonen et al., 2011) (FIG. 8A). The secretomes showed a pH optimum of ˜7 for reaction with AFEX-SG and a pH optimum of ˜8 for reaction with IL-SG. Moreover, these secretomes retained greater than 60% of maximal activity over the ranges of pH 4.5 to 8.0, and pH 7.0 to 8.0, respectively (FIG. 8B). These optimal pH values are considerably higher than observed for Spezyme CP.

Example 9 ActE Produces Cellobiose as the Primary Extracellular Product of Cellulose Utilization

The isolated ActE secretomes contained substantial ability to release reducing sugars from pure polysaccharides. Cellobiose accounted for ˜95% of soluble sugar released from pure cellulose and glucose represented the remainder; cellotriose and cellobionic acid were not detected. Neither cellobiosidase nor β-glucosidase was detected in the ActE secretome. Thus ActE produces cellobiose as the primary extracellular product of cellulose utilization and also grows vigorously on this. Dominance of cellobiose may help to channel cellulolytic activity to only a subset of the Sirex community. Since genes encoding cellobiose oxidase and cellobiose dehydrogenase (Eastwood et al., 2011; Langston et al., 2011) were not present in ActE, biological reduction systems for the CBM33 proteins may be provided by other members of the Sirex community, in analogy to that described for the heterologous complex of T. aurantiacus GH61 and Humicola insolens cellobiose dehydrogenase (Langston et al., 2011).

Example 10 Enzymatic Activity of the ActE Secretome can be Improved by Adding One or More Enzymes from other Organisms or Sources

In the ActE secretome, enzymes SACTE_0236, SACTE_0237, and SACTE_3717 (GH48, GH6, and GH9, respectively) showed decreases in content of the native forms over a 24 h period, and SACTE_0236 and SACTE_0237 were converted into ˜50 kDa fragments (FIG. 24). SACTE_0359 (CBM33) also showed a time-dependent decrease. The reactions could be slowed but not eliminated by addition of phenylmethylsulfonyl fluoride (1 mM), a possible inhibitor of serine proteases (Turini et al., 1969), but not by EDTA (10 mM), a possible inhibitor of metalloproteases (Trop and Birk, 1970).

SACTE_5668, a serine protease, was detected in all pure polysaccharide secretomes (FIG. 3), while another metallopeptidase, SACTE_3389 annotated as peptidase M24B, X-Pro dipeptidase/aminopeptidase P, was detected in all secretomes at low level (0.026%). The protease SACTE_5530 (peptidase S1/S6, chymotrypsin/Hap, 0.1%) was also present in all polysaccharide and biomass samples. The proteases SACTE_5668 (annotated secreted peptidase, 0.3%) and SACTE_4231 (serine/cysteine peptidase, trypsin-like, 0.039%) were also detected in all pure polysaccharide secretomes, and the protease SACTE_6303 (serine/cysteine peptidase, trypsin-like, 0.039%) was also present in all biomass samples. Elimination of one or more of these proteases may impart stabilization of the enzymatic activity in the secreted proteome.

Addition of CelLcc_CBM3a (SEQ ID Nos:63 and 64), an engineered exoglucanase (FIG. 22) that produces cellobiose with low specific activity alone (FIG. 21), gave a synergistic increase in the activity of the ActE cellulose secretome. This result demonstrates the potential for heterologous supplementation of the ActE secretome to improve its performance by replacing an enzyme activity that is lost to proteolysis.

Example 11 ActE Cellulolytic Activity Requires a Minimal Set of Enzymes

When the ActE secretome obtained from growth on cellulose was fractionated by ion exchange chromatography (FIG. 7), several fractions were obtained that could be tested in unary, binary, ternary and quaternary combinations for reconstitution of cellulose hydrolysis and other enzymatic activities (FIG. 25). SDS PAGE and LC-MS/MS analysis showed that these fractions contained the following polypeptides in the approximate weight percentages: fraction 1, SACTE_3159 (CBM33/CBM2 oxidative endocellulase, 95%) and SACTE_4738 (GH16 β-1,3 endoglucanase, 5%); fraction 2, SACTE_0237 (GH6 non-reducing end exocellulase, 60%), SACTE_0482 (GH5 endocellulase, 25%), SACTE_4755 (GH64 β-1,3 glucanase, 10%) and SACTE_3159 (oxidative endocellulase, <5%); and fraction 3, SACTE_0236 (GH48 reducing end exocellulase, 75%), SACTE_3717 (GH9 endocellulase, 20%) and SACTE_5457 (GH46 chitinase, 5%). These results demonstrate that SACTE_3159 (oxidative endocellulase) provides a complementary activity to SACTE_0482 and SACTE_3717 (hydrolytic endocellulolytic activity). Evidently, the oxidative reaction provides breaks in the cellulose strands that can be readily used by non-reducing and reducing end exocellulases also present in the secretome to processively deconstruct the polymeric material.

According to the current understanding of reactions required for hydrolysis of crystalline cellulose, SACTE_3159 (CBM33/CBM2 oxidative endocellulase), SACT_0482 (GH5), and SACTE_3717 provide endocellulolytic activities, while SACTE_0237 (GH6) provides non-reducing end exocellulase reaction and SACTE_0236 (GH48) provides reducing end exocellulase activity.

FIG. 16 shows that the secretome contains beta-1,3 endoglucanase activity. The majority of this activity corresponds to the fractions containing SACTE_4738 and SACTE_4755. These enzymes hydrolyze callose, a cellulose-like material that is typically produced by plants in respond to wounding by invasive insects and other trauma.

The proteins described here constitute a naturally evolved and matched set specialized for the hydrolysis of cellulosic substrates.

Example 12 ActE Mannanase Specific Activity Increases as Mannanase Molecular Weight Decreases

FIG. 26 shows that the mannanase activity present in the ActE secretome is associated with fractions containing various naturally truncated variants of SACTE_2347 (GH5) with molecular weights of ˜57, ˜45, and ˜37 kDa. Fractions F9 through F1 from ion exchange chromatographic separation of the ActE secretome were examined for mannan-deconstruction activity by Zymogram assay. The basis of the Zymogram assay is as follows: Congo Red stain interacts with the polysaccharide fraction (mannan) incorporated into the gel and imparts a red color. When an enzyme's activity hydrolyzes the mannan, the interaction of Congo Red with the polysaccharide is broken and the gel takes on a dark grey appearance. Of note, the strongest mannanase activity was observed in fraction F1, which primarily contains the 37 kDa truncated variant. Corresponding to the definition of specific activity given above, the 37 kDa variant has an ˜35% increase in specific activity relative to the 57 kDa variant. This provides a naturally produced example of how size reduction may contribute to increased specific activity of enzymes.

Example 13 Recombination of ActE Secretome Fractions Provides Synergistic Cellulolytic Activity

FIG. 25 shows synergy of reaction obtained by recombining fractions obtained from ion exchange fractionation. In FIG. 25A, reactions were obtained from combinations of the fractions indicated by stars in FIG. 27 and FIG. 28. Combination of fractions E5 (oxidative endocellulase) and E11 (hydrolytic endo- and exocellulases) gave a ˜30% increase in product yield over that expected from the arithmetic sum of reactions of E5 and E11 alone, i.e., synergy in reaction. Combination of fractions E5, E11 and F10 (hydrolytic endo- and exocellulases) gave ˜60% increase in reactivity. In FIG. 25B, reactions were obtained from recombining fractions shown in FIG. 16. Titration of fraction B1 (full-length oxidative endocellulase) into D15 (hydrolytic endo- and exocellulases) shows an optimal reactivity at ˜1:1 ratio of proteins from the two fractions, while an excess of B1 relative to D15 causes decrease in reaction because of depletion of required exocellulase activities. Titration of fraction C4 (truncated oxidative endocellulase and beta-1,3-endocellulase) with D15 gave maximal stimulation (62% increase) at an 80:20 proportion. These results indicate both forms of oxidative endocellulase SACTE_3159 are catalytically active, with the smaller form providing a higher synergistic response, again corresponding to a specific activity increase associated with size minimization.

Example 14 The Function of ActE Xylanases can be Assigned by Functional Assay of Proteins Produced by Using Cell-free Translation

FIG. 29 shows that both of the xylanases identified in the fractions of ActE secretomes obtained from ion exchange chromatography can also be expressed using cell-free translation and demonstrated to be xylanases by catalytic activity assays. These proteins are SACTE_0265 and SACTE_0358. Other proteins that are not secreted were successfully expressed (SACTE_2548, SACTE_2286, SACTE_437) as control proteins, and as expected from their predicted intracellular localization, none of these controls exhibited xylanase activity. The negative result with the control proteins also demonstrates that the wheat germ extract used for cell-free translation of novel cellulolytic enzymes does not have an endogenous xylanase activity, as established in US Patent Application Publication No.: US2010/037094 (Fox and Elsen).

Example 15 Total Protein Secreted by ActE can be Increased

A minimal set of enzymes for biomass deconstruction can be defined by combining the additional enzymes whose expression is elicited during growth on biomass (Table 1) with enzymes uniquely expressed during growth on cellulose and xylan.

Besides assembling the proper enzymatic constituents, the level of total protein secreted is an important biotechnological constraint for industrial enzyme production. FIG. 30 shows the non-optimized level of secreted protein obtained from growth of ActE on different biomass substrates. By use of lignocellulosic substrates for growth, secreted protein levels up to 0.25 g per liter of culture medium can be readily obtained. Growth on non-polymeric substrates such as cellobiose does not elicit a secreted protein response. FIG. 15, FIG. 16 and Table 1 indicate that the twin-arginine pathway (Tat) is used during growth, thus identifying this pathway as playing a key role in the secretion of enzymes required for extracellular deconstruction of biomass polysaccharides (Natale et al., 2008; Chater et al., 2010). Methods to increase the titer of secreted proteins are known, and have been highly effectively when applied to Streptomyces and other organisms (Cereghino et al., 2002; Zhang et al., 2006; Nijland and Kuipers, 2008; Chater et al., 2010; Schuster and Schmoll, 2010). These established methods can be applied to ActE to obtain more concentrated secretome preparations.

Example 16 ActE Enzymatic Activity Corresponds with Optimal Growth Conditions of Fermentation Organisms

FIG. 31 shows the temperature versus activity profile for ActE secretomes for reaction with cellulose, xylan and mannan. These profiles are well matched to the growth optima range for mesophilic fermentation organisms such as Saccharomyces cerevisiae, Zymomonas mobilis, Escherichia coli or others (Jarboe et al., 2010; Peralta-Yahya and Keasling, 2010), which are widely used for ethanol production from sugar hydrolysates. These hydrolysates are produced from biomass by the enzymatic action of highly cellulolytic secretomes, such as those described here from ActE. These optima are also well matched with the conditions found in the rumen, where the efficiency of conversion of animal feed, which is a biomass material, can be improved by addition of enzymes.

FIG. 32 shows the pH versus activity profiles for ActE secretomes for reaction with cellulose, xylan and mannan. These profiles are well matched to the growth optima range for fermentation organisms such as S. cerevisiae, Z. mobilis, E. coli or other organisms (Jarboe et al., 2010; Peralta-Yahya and Keasling, 2010) which are widely used for ethanol production from sugar hydrolysates such as might be produced from biomass by a highly cellulolytic secretome, such as those described here from ActE. These optima are also well matched with the conditions found in the rumen, where the efficiency of conversion of animal feed, which is a biomass material, can be improved by addition of enzymes. The ActE secretome retains high specific activity (>80% of maximal) at pH 7, which closely approximates that of the rumen. Sectetomes from fungi such as T. reesei are considerably less active at neutral pH, rendering them less effective at neutral pH.

The high cellulolytic capacity of ActE, and its corresponding secretomes, coupled with the temperature and pH optima described above permit assembly of two-part systems to effect the simultaneous deconstruction of biomass and fermentation to fuels.

Example 17 ActE Induction in Medium Containing Various Percentages of Cellulose

To determine ActE's growth profile on cellulose as a carbon source ActE was grown in M63 media plus 5 g/L carbon. The carbon source ratio was adjusted from 100% cellulose to 100% glucose, total carbon in each culture was equal. Cells were grown for 6 days at 30 degrees. Supernatant was harvested, filtered, and separated by 4-20% SDS-PAGE. Results suggest that ActE is induced in media containing as little as 20% cellulose, with optimal induction in medium containing between 80%-100% cellulose (FIG. 33).

Example 18 Discussion

The work presented here provides the first genome-wide insight into how an aerobic microbe deconstructs polysaccharides. ActE achieves efficient utilization of cellulose by a simple combination of well-understood hydrolytic reactions with newly identified oxidative reactions. The two required exoglucanases are each encoded by a single gene, which also represents the only example of their respective GH families in the genome. The proteins encoded by these genes provide reactions that are complementary to the reactions of other enzymes in the secretome, and provide cellobiose as the major product of reaction. We have discovered that many of the highly abundant enzymes secreted by ActE during growth on cellulose have reduced size relative to their orthologs from closely related organisms. This novel finding suggests natural evolution to improve specific activity has already occurred in ActE in response to growth in the highly specialized insect association. Additional specializations of ActE were identified by demonstrating the secretion of a unique set of proteins in response to biomass. In addition, this work defines how simple new combinations of improved biomass deconstruction enzymes can be assembled according to the propensities of the naturally evolved system.

The present work also indicates that insect-associated microbes such as ActE are important contributors to the vigorous attack on biomass by insects. The ‘highly invasive’ designation given to Sirex has been generally attributed to the combined action of wasp and fungus (Tabata and Abe, 2000; Bergeron et al., 2011). Species convergence is now recognized in the microbial communities associated with insects (Suen et al., 2010; Hulcr et al., 2011). Given the ubiquitous presence of Streptomycetes in these communities, the enzymatic properties described here also contribute a potential risk to pine forests, including those used for industrial purposes.

The invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.

REFERENCES

-   -   1. Baldrian, P. & Valaskova, V. FEMS Microbiol Rev 32, 501-521         (2008).     -   2. Cantarel, B. L. et al. Nucleic Acids Res 37, D233-238 (2009).     -   3. Schuster, A. & Schmoll, M. Appl Microbiol Biotechnol 87,         787-799 (2010).     -   4. Crawford, D. L. Appl Environ Microb 35, 1041-1045 (1978).     -   5. Goodfellow, M. & Williams, S. T. Annu Rev Microbiol 37,         189-216 (1983).     -   6. McCarthy, A. J. & Williams, S. T. Gene 115, 189-192 (1992).     -   7. Schlatter, D. et al. Microb Ecol 57, 413-420 (2009).     -   8. Wachinger, G., Bronnenmeier, K., Staudenbauer, W. L. &         Schrempf, H. Appl Environ Microb 55, 2653-2657 (1989).     -   9. Ishaque, M. & Kluepfel, D. Can J Microbiol 26, 183-189         (1980).     -   10. Semedo, L. T. et al. Int J Syst Evol Microbiol 54,         1323-1328, (2004).     -   11. Schlochtermeier, A., Walter, S., Schroder, J., Moorman, M. &         Schrempf, H. Mol Microbiol 6, 3611-3621 (1992).     -   12. Walter, S. & Schrempf, H. Appl Environ Microb 62, 1065-1069         (1996).     -   13. Forsberg, Z. et al. Protein Science 20, 1479-1483 (2011).     -   14. Bignell, D. E., Anderson, J. M. & Crosse, R. Fems Microbiol         Ecol 85, 151-159 (1991).     -   15. Pasti, M. B. & Belli, M. L. Fems Microbiol Lett 26, 107-112         (1985).     -   16. Pasti, M. B., Pometto, A. L., Nuti, M. P. & Crawford, D. L.         Appl Environ Microb 56, 2213-2218 (1990).     -   17. Schafer, A. et al. J Appl Bacteriol 80, 471-478 (1996).     -   18. Adams, A. S. et al. ISME Journal 5, 1323-1331 (2011).     -   19. Bergeron, M. J. et al. Fungal Biol 115, 750-758 (2011).     -   20. Kukor, J. J. & Martin, M. M. Science 220, 1161-1163 (1983).     -   21. Lynd, L. R., Weimer, P. J., van Zyl, W. H. &         Pretorius, I. S. Microbiol Mol Biol Rev 66, 506-577 (2002).     -   22. Deboy, R. T. et al. J Bacteriol 190, 5455-5463 (2008).     -   23. Riederer, A. et al. Appl Environ Microb 77, 1243-1253         (2011).     -   24. Marushima, K., Ohnishi, Y. & Horinouchi, S. J Bacteriol 191,         5930-5940 (2009).     -   25. Walter, S. & Schrempf, H. Mol Gen Genet 251, 186-195 (1996).     -   26. Deng, Y. & Fong, S. S. Appl Environ Microb 76, 2098-2106         (2010).     -   27. Li, C. et al. Bioresour Technol 102, 6928-6936 (2011).     -   28. Tolonen, A. C. et al. Mol Syst Biol 6, 461 (2011).     -   29. Hyatt, D. et al. BMC Bioinformatics 11, 119 (2010).     -   30. Rutherford, K. et al. Bioinformatics 16, 944-945 (2000).     -   31. Medema, M. H. et al. Nucleic Acids Res 39, W339-346 (2011).     -   32. Shannon, P. et al. Genome Res 13, 2498-2504 (2003).     -   33. Miller, G. L. Anal Chem 31, 426-428 (1959).     -   34. Merino and Chemy, Adv. Biochem. Eng. Biotechnol. 108:95-120,         2007     -   35. Bayer et al., Chem. Rec. 8:364-377, 2008     -   36. Wilson, Curr. Opin. Microbiol. 14:259-263, 2011     -   37. Vuong and Wilson, Biotechnol. Bioeng. 107:195-205, 2010     -   38. Scharf et al., PLoS One 6: e21709, 2011     -   39. Luyten et al., App. Environ. Microbiol. 72:412-417, 2006     -   40. Hess et al., Science 331:463-467, 2011     -   41. Schlochtermeier et al., App. Environ. Microbiol.         58:3240-3248, 1992     -   42. Wilson, Crit. Rev. Biotechnol. 12:45-63; 1992     -   43. Langston et al., App. Environ. Microbiol. 77:7007-7015, 2011     -   44. Quinlan et al., PNAS 108:15079-15084, 2011     -   45. Vaaje-Kolstad et al., Science 330:219-222, 2010     -   46. Klepzig et al., Environ. Entomol. 38: 67-77, 2009     -   47. Teather R M, Wood P J (1982) Appl Environ Microbiol         43:777-780     -   48. Bradford, Anal. Biochem. 72:248-254 (1976)     -   49. Anne and Van Mellaert. FEMS Microbiol. Lett., 114; 121-8         (1993)     -   50. Saloheimo and Pakula, Microbiology, Epub date 2011 Nov. 5     -   51. Wood et al., Biotechnol. Bioeng. 55:547-55 (1997)     -   52. Balan et al., Meth. Mol. Biol. 581:61-77; 2009     -   53. Makino et al., Meth. Microbiol. 607:127-134, 2010     -   54. Blommel et al., Meth. Mol. Biol. 498:55-73, 2009     -   55. Galm et al., J. Nat. Prod. 74:526-536, 2011;     -   56. Susi et al. J. Environ. Manage. 92:1681-1689, 2011     -   57. Balan et al., Meth. Mol. Biol. 581:61-77, 2009     -   58. Chundawat et al., J. Am. Chem. Soc. 133:11163-11174, 2011     -   59. Cheng et al., Biomacromol. 12:933-941, 2011     -   60. Wang et al., Biotechnol. Progress 25:1086-1093, 2009     -   61. Tian et al., Biotechnol. Progress 27: 419-427, 2011     -   62. Altschul et al., J. Mol. Biol. 215:403-410, 1990     -   63. Herpoel-Gimbert et al., Biotechnol. Biofuels 1:18; 2008     -   64. Raman et al., PLoS One 4:e5271, 2009     -   65. Chen and Wilson, J. Bacteriol. 189:6260-6265, 2007     -   66. Eastwood et al., Science 333:762-765, 2011     -   67. Langston et al., App. Environ. Microbiol. 77:7007-7015, 2011     -   68. Turini et al., J. Pharmacol. Exper. Therapeu. 167:98-104,         1969     -   69. Trop and Birk, Biochem. J. 27:419-427, 1970     -   70. Natale et al., Biochim. Biophys. Acta 1778:1735-1756, 2008     -   71. Chater et al., FEMS Microbiol. Rev. 34:171-198, 2010     -   72. Cereghino et al., Curr. Opin. Biotechnol. 13:329-332, 2002     -   73. Zhang et al., Nat. Biotechnol. 24:100-104, 2006     -   74. Nijland and Kuipers, Recent Pat. Biotechnol. 2:79-87, 2008     -   75. Jarboe et al., J. Biomed. Biotechnol. 761042, 2010     -   76. Peralta-Yahya and Keasling, Biotechnol. J. 5:147-162, 2010     -   77. Tabata and Abe, Mycoscience 41:585-539, 2000     -   78. Suen et al., PLoS Gen. 6: e1001129, 2010     -   79. Hulcr et al., Micro. Ecol. 61:759-768, 2011     -   80. Kestler et al., BMC Bioinform. 9:67, 2008 

We claim:
 1. A method for digesting a non-wood biomass lignocellulosic material, wherein the method comprises: recombinantly expressing in a non-native microbial host cell SActE_0237 (GH6) (SEQ ID NO: 1), SActE_0265 (GH10) (SEQ ID NO: 5) and SActE_0236 GHQ48) (SEQ ID NO: 2), isolating SActE_0237 (GH6) (SEQ ID NO: 1), SActE_0265 (GH10) (SEQ ID NO: 5) and SActE_0236 (GH48) (SEQ ID NO: 2) from the host cell, and exposing the non-wood biomass lignocellulosic material to a sufficient amount of a composition comprising the isolated SActE_0237, SActE_0265, and SActE_0236, wherein the exposed lignocellulosic material is at least partially digested.
 2. The method of claim 1, wherein the microbial host is selected from the group consisting of Streptomyces lividans, Trichoderma reesei, and Escherichia coli.
 3. The method of claim 1, wherein the method additionally comprises recombinantly expressing in a microbial host at least one member selected from the group consisting of SActE_0357 (CE4) (SEQ ID NO:7), SActE_0358 (GH11) (SEQ ID NO:8), SActE_1310 (PL3) (SEQ ID NO:9), SActE_3717 (GH9) (SEQ ID NO:10), SActE_4638 (SEQ ID NO:11), SActE_4738 (GH16) (SEQ ID NO:12), SActE_4755 (GH64) (SEQ ID NO:13), SActE_5457 (GH46) (SEQ ID NO:14), SActE_5647 (GH87) (SEQ ID NO:15), and SActE_5978 (PL1) (SEQ ID NO:16) to produce a composition comprising isolated SActE 0237, SActE_0265, SActE_0236, and the at least one member, and exposing the at least 85% non-wood biomass lignocellulosic material to a sufficient amount of the composition comprising isolated SActE_0237, SActE_2065, SActE_0236, and the at least one member.
 4. A method for digesting a non-wood biomass lignocellulosic material, wherein the method comprises: genetically modifying an ActE strain to overexpress SActE_0237 (GH6) (SEQ ID NO:1). SActE_0265 (GH10) (SEQ ID NO:5), and SActE_0236 (GH48) (SEQ ID NO:2), and exposing the non-wood biomass lignocellulosic material to the genetically modified ActE strain, wherein the exposed lignocellulosic material is at least partially digested.
 5. The method of claim 4, wherein the ActE strain is further genetically modified to overexpress least one member selected from the group consisting of SActE_0357 (CE4) (SEQ ID NO:7), SActE_0358 (GH11) (SEQ ID NO:8), SActE_1310 (PL3) (SEQ ID NO:9), SActE_3717 (GH9) (SEQ ID NO:10), SActE_4638 (SEQ ID NO:11), SActE_4738 (GH16) (SEQ ID NO:12), SActE_4755 (GH64) (SEQ ID NO:13), SActE_5457 (GH46) (SEQ ID NO:14), SActE_5647 (GH87) (SEQ ID NO:15), and SActE_5978 (PL1) (SEQ ID NO:16).
 6. A method for enzymatically pretreating agricultural crop materials for consumption by ruminant animals, wherein the method comprises: recombinantly expressing in a non-native microbial host cell SActE_0237 (GH6) (SEQ ID NO:1), SActE_0265 (GH10) (SEQ ID NO:5) and SActE_0236 (GH48) (SEQ ID NO:2), harvesting said host cells, and exposing agricultural crop materials for consumption by ruminant animals to the host cells, wherein the agricultural crop materials are at least partially digested.
 7. The method of claim 6, wherein the host cell is Saccharomyces cerevisiae or Escherichia coli. 